Journal articles: 'PCR methodologies'

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Martínez, Ana Laura, Freda Anderson, Facundo Quiroz, Antonio Garayalde, Ignacio Erreguerena, Lorena Armando, Norma Huguet, and Alicia Carrera. "Methodologies for Plasmopara halstedii Research." Helia 42, no. 71 (November 18, 2019): 173–86. http://dx.doi.org/10.1515/helia-2019-0013.

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Abstract The objective of this work was to find practical procedures to overcome methodological drawbacks encountered during studies on sunflower downy mildew. Techniques for recovering living isolates of Plasmopara halstedii from the field and for the preservation of infected leaf samples for further molecular analysis were developed. A Polymerase Chain Reaction (PCR)-based test for the detection of P. halstedii in sunflower leaves and a method to remove azoxystrobin from fungicide-treated seeds are proposed. In situ-inoculations of pre-germinated seeds allowed the recovery of living isolates from the field. Three sample-preservation methods were evaluated (silica, heating and lyophilization) resulting in high yield and quality of the DNA extract. It was detected the presence of the pathogen in symptomless leaves through PCR using molecular markers based on expressed sequence tags. A treatment using sodium hypochlorite is recommended for the removal of azoxystrobin from fungicide treated seeds.

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Twin, J., N. Taylor, S. M. Garland, J. S. Hocking, J. Walker, C. S. Bradshaw, C. K. Fairley, and S. N. Tabrizi. "Comparison of Two Mycoplasma genitalium Real-Time PCR Detection Methodologies." Journal of Clinical Microbiology 49, no. 3 (January 5, 2011): 1140–42. http://dx.doi.org/10.1128/jcm.02328-10.

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Múrtula, Raquel, Elena Soria, M. Adela Yáñez, and Vicente Catalán. "Proficiency testing schemes for the assessment of Legionella PCR methodologies." Accreditation and Quality Assurance 17, no. 4 (June 9, 2012): 431–37. http://dx.doi.org/10.1007/s00769-012-0903-5.

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Migliaro, Daniele, Giacomo Morreale, Massimo Gardiman, Sara Landolfo, and Manna Crespan. "Direct multiplex PCR for grapevine genotyping and varietal identification." Plant Genetic Resources 11, no. 2 (December 5, 2012): 182–85. http://dx.doi.org/10.1017/s1479262112000433.

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Grapevine cultivar identification is based mainly on two complementary methodologies: microsatellite (simple sequence repeat (SSR)) DNA analysis and traditional ampelography. Here, we report a direct multiplex PCR approach that allows the simultaneous amplification of 11 SSR loci from crude samples, i.e. bypassing DNA extraction, by using an engineered DNA polymerase improved to tolerate plant PCR inhibitors. Many different plant tissues were successfully amplified: leaf, root, wood, berry flesh and skin, stalk and must, from wine and table grape varieties, and rootstocks. The direct multiplex PCR that we propose is quicker and cheaper than the methodologies used until now, and provides accurate results. Thus, SSR DNA analysis becomes economically more accessible to a larger number of potential users in addition to research institutes.

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Herrero, Beatriz, Fátima C. Lago, Juan M. Vieites, and Montserrat Espiñeira. "Authentication of swordfish (Xiphias gladius) by RT–PCR and FINS methodologies." European Food Research and Technology 233, no. 2 (June 5, 2011): 195–202. http://dx.doi.org/10.1007/s00217-011-1502-0.

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Linars, Artis, Ivars Silamikelis, Dita Gudra, Ance Roga, and Davids Fridmanis. "OverFlap PCR: A reliable approach for generating plasmid DNA libraries containing random sequences without a template bias." PLOS ONE 17, no. 8 (August 8, 2022): e0262968. http://dx.doi.org/10.1371/journal.pone.0262968.

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Over the decades, practical biotechnology researchers have aimed to improve naturally occurring proteins and create novel ones. It is widely recognized that coupling protein sequence randomization with various effect screening methodologies is one of the most powerful techniques for quickly, efficiently, and purposefully acquiring these desired improvements. Over the years, considerable advancements have been made in this field. However, developing PCR-based or template-guided methodologies has been hampered by resultant template sequence biases. Here, we present a novel whole plasmid amplification-based approach, which we named OverFlap PCR, for randomizing virtually any region of plasmid DNA without introducing a template sequence bias.

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Goh, Su Kah, Ashan Musafer, Tom Witkowski, Vijayaragavan Muralidharan, Christopher Christophi, Hongdo Do, and Alexander Dobrovic. "Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms." Clinical Chemistry 62, no. 7 (July 1, 2016): 1012–19. http://dx.doi.org/10.1373/clinchem.2016.256388.

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Abstract BACKGROUND The quantification of genomic chimerism is increasingly recognized for its clinical significance after transplantation. Before the measurement of chimerism, accurate genotyping of genetic polymorphisms for informative alleles that can distinguish donor DNA from recipient DNA is essential. The ease of allelic discrimination of small deletion and insertion polymorphisms (DIPs) makes DIPs attractive markers to track chimerism. Current methodologies for the genotyping of DIPs are largely based on “open-tube” approaches. “Closed-tube” approaches involving no or minimal post-PCR handling are preferred. We compared 3 distinct methodologies to determine an optimal platform for DIP genotyping. METHODS Genomic DNA from 19 normal individuals was genotyped for 6 small biallelic DIPs using high-resolution melting analysis (HRMA), probe-free droplet digital PCR (ddPCR), and microfluidic electrophoresis of PCR products. For HRMA, 3 different platforms were compared. RESULTS Our newly developed probe-free ddPCR approach allowed the genotype of each DIP to be determined by fluorescence intensity based on amplicon size. Microfluidic electrophoresis also allowed genotypes to be determined by amplicon size. HRMA assays allowed the genotype of each DIP to be determined by melting profile. Genotyping results were concordant between the 3 methodologies. HRMA was the most readily performed methodology and was robust across 3 separate HRMA-capable platforms. CONCLUSIONS We demonstrated the effectiveness of probe-free ddPCR to accurately genotype small biallelic DIPs. Nevertheless, HRMA proved to be the optimal approach for genotyping small DIPs because closed-tube approaches are preferred owing to rapid and less laborious workflows and least risk of PCR contamination.

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Reynolds, Kelly A., Kimberly Roll, Roger S. Fujioka, Charles P. Gerba, and Ian L. Pepper. "Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies." Canadian Journal of Microbiology 44, no. 6 (June 1, 1998): 598–604. http://dx.doi.org/10.1139/w98-040.

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The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase - polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.Key words: enteroviruses, RT-PCR, cell culture, marine waters.

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Patterson, Thomas F., and J. Peter Donnelly. "New Concepts in Diagnostics for Invasive Mycoses: Non-Culture-Based Methodologies." Journal of Fungi 5, no. 1 (January 17, 2019): 9. http://dx.doi.org/10.3390/jof5010009.

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Non-culture-based diagnostics have been developed to help establish an early diagnosis of invasive fungal infection. Studies have shown that these tests can significantly impact the diagnosis of infection in high risk patients. Aspergillus galactomannan EIA testing is well-recognized as an important adjunct to the diagnosis of invasive aspergillosis and can be detected in serum, bronchoalveolar lavage and other fluids. Galactomannan testing used along with PCR testing has been shown to be effective when integrated into care paths for high risk patients for both diagnoses and as a surrogate marker for outcome when used in serial testing. Beta-d-glucan assays are non-specific for several fungal genera including Aspergillus and Candida and in high risk patients have been an important tool to augment the diagnosis. Lateral flow technology using monoclonal antibodies to Aspergillus are available that allow rapid testing of clinical samples. While standard PCR for Candida remains investigational, T2 magnetic resonance allows for the rapid diagnosis of Candida species from blood cultures. Aspergillus PCR has been extensively validated with standardized approaches established for these methods and will be included in the diagnostic criteria in the revised European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSG) definitions. Finally, these non-culture-based tests can be used in combination to significantly increase the detection of invasive mycoses with the ultimate aim of establishing an early diagnosis of infection.

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Romero, Atocha, Miguel Angel Molina-Vila, Eloisa Jantus-Lewintre, Amelia Insa, Patricia Cruz, Ana Collazo, Javier Perez Altozano, et al. "Comprehensive cross-platform comparison of methodologies for noninvasive EGFR mutation testing: Results of the RING observational trial." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e21518-e21518. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21518.

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e21518 Background: Several platforms for non-invasive EGFR testing are currently used in the clinical setting, with sensitivities ranging from 30 to 100%. Comparison studies in prospective cohorts remain limited and reports evaluating mutant allelic fractions (MAFs) are particularly scarce. The RING observational trial (ClinicalTrials.gov identifier NCT03363139) was designed to comprehensively analyze the concordance between methodologies for EGFR mutation detection in blood. Methods: Seventy-two EGFR mutant NSCLC patients were enrolled in the trial. Plasma samples were prospectively collected at progression to first line Tyrosine Kinase Inhibitor and tested for EGFR mutations by 7 methodologies; cobas EGFR Mutation Test v2, Therascreen EGFR Plasma RGQ PCR Kit, QuantStudio 3D Digital PCR System, a 5-nuclease real-time PCR assay in presence of PNA, OncoBEAM EGFR and NGS with two different gene panels, Ion Torrent Oncomine and GeneRead QIAact Lung DNA UMI Cancer Panel. Results: The agreement between all methodologies for was almost perfect for the detection of deletions in exon 19 (K = 0.86; 95%CI: 0.76-0.96) and substantial for exon 21 point mutations (K = 0.76; 95%CI: 0.63-0.89). Regarding the p.T790M resistance mutation, concordance was lower but still substantial (K = 0.68; 95%CI: 0.57-0.79). If only NGS-based technologies were considered, the agreement was almost perfect for sensitizing mutations and substantial for the resistance mutation (K = 0.84; 95%CI: 0.68-1.00, K = 0.86; 95%CI: 0.69-1.00 and K = 0.77; 95%CI: 0.60-0.95 for exon 19, exon 21 and p.T790M, respectively). Most discordant samples between methodologies had mutant allele fractions (MAFs) ≤0.5%. Sensitizing mutations were always present at higher MAFs than concomitant p.T790M, explaining the lower concordance observed for this variant. MAFs obtained by different methodologies showed an excellent reproducibility (intraclass correlation coeficients 0.85-0.97). Similarly, Passing–Bablok regression analysis showed a high correlation between methodologies when assessing MAFs. Conclusions: Our results support the use of liquid biopsies for non-invasive EGFR testing in the clinical setting and highlight the need to systematically report MAFs.

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Acosta Soto, Lucrecia, Ana Belén Santísima-Trinidad, Fernando Jorge Bornay-Llinares, Marcos Martín González, José Antonio Pascual Valero, and Margarita Ros Muñoz. "Quantitative PCR and Digital PCR for Detection ofAscaris lumbricoidesEggs in Reclaimed Water." BioMed Research International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/7515409.

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The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control.Ascaris lumbricoidesis one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts ofA. lumbricoideseggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as oneA. lumbricoidesegg equivalent, while dPCR can detect DNA concentrations as low as fiveA. lumbricoidesegg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20A. lumbricoideseggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks.

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Zuluaga, Alejandra, Karen Arango Bustamante, Diego H. Caceres, Zilpa A. Sánchez Quitian, Verónica Velásquez, Beatriz L. Gómez, Claudia M. Parra Giraldo, et al. "Concordance analysis between different methodologies used for identification of oral isolates of Candida species." Colombia Médica 49, no. 3 (September 1, 2018): 193–200. http://dx.doi.org/10.25100/cm.v49i3.3774.

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Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

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Martinez, Rafael C. R., Sílvio A. Franceschini, Maristela C. Patta, Silvana M. Quintana, Álvaro C. Nunes, João L. S. Moreira, Kingsley C. Anukam, Gregor Reid, and Elaine C. P. De Martinis. "Analysis of Vaginal Lactobacilli from Healthy and Infected Brazilian Women." Applied and Environmental Microbiology 74, no. 14 (May 23, 2008): 4539–42. http://dx.doi.org/10.1128/aem.00284-08.

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ABSTRACT Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H2O2-producing lactobacilli were not associated with protection against VVC.

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Rossi, Giovanni, Mariella Minervini, Angelo Michele Carella, Chiara de Waure, Lorella Melillo, Giovanni Francesco D'Arena, Nicola Cascavilla, and Gina Zini. "Comparison of the Performance of Multiparameter Flow-Cytometry and wt1-RNA Levels in Detecting Minimal Residual Disease and Predicting Relapse in Patients with Acute Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 2507. http://dx.doi.org/10.1182/blood.v118.21.2507.2507.

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Abstract Abstract 2507 Introduction Despite a high remission rate, a significant number of patients with acute myeloid leukemia (AML) relapse (Lowenberg et al. 2003). Thus, the evaluation of minimal residual disease (MRD) in AML is an important strategy to better identify high risk patients. Most sensitive is the molecular polymerase chain reaction (PCR) methodology but its applicability is restricted to subgroups of AML with leukemia-specific molecular targets (e.g. aml1-eto, cbfb-myh11, mll, flt3). However, these cases comprise only 40–50% of all AML cases (Kern et al. 2004). Detection of aberrant phenotypes by multiparameter flow cytometry (MFC)(San Miguel et al. 2001, Al Mawali et al. 2009) and quantification of wt1-RNA levels (Cilloni et al.2008) provide excellent options for MRD monitoring in AML. In the present study we investigated patients with AML withouth leukemia- specific genetic alterations. In this subgroup, in order to realize the methodology that better identify high risk patients, the performance of both MFC and wt1 expression (RQ-PCR) was evaluated. Then, if threshold values defining a presence of MRD for both of methodologies are predictors of poor prognosis were also evaluated. Patients and methods Fresh bone marrow samples from 74 AML patients were obtained at diagnosis between January 2008 and May 2011. CR was achieved from 59 patients. Of these patients, 23 did not show genetic alterations and were studied for MRD with both MFC and wt1 expression (RQ-PCR) at different time points: after induction therapy (T1), after consolidation therapy (T2) and every 3 months during follow-up. The immunophenotypic analysis was performed using two panels of markers in a six-color combinations in order to correctly identify leukemia –associated immunophenotypes (LAIP). The follow MoAb CD45/CD34/CD117/CD15 and CD45/CD34/CD64/14 were combined with specific myeloid and lymphoid markers respectively. RQ- PCR to test wt1 expression was made according to the standardized and quality-controlled method (Willasch et al. 2009). The obtained wt1 copy numbers were normalized with respect to the ABL transcripts. Results An analysis of sensitivity, specificity, predictive values(PV), likelihood ratio(LR), Receiver Operating characteristic Curve (ROC) and the Area Under the Curve (AUC) for both MCF and RQ-PCR of wt1 were performed. MFC showed higher sensitivity than RQ-PCR at each time point (80% vs 70% at T1) but specificity of RQ-PCR was always superior to MFC, particularly at T2 (76,9% vs 53,8%). Although both methodologies showed comparable LR+ values at each time point, a better LR− for MFC analysis was found (LR+: 1,73 vs 1,82; LR−:0,37 vs 0,48 at T1).However both methodologies showed low positive PV (57,1 vs 58,3 at T1). AUC and 95% confidence intervals demonstrated a moderate accuracy for both MFC and RQ-PCR [0,715 (0,499–0,932) vs 0,713 (0,506–0,940) at T1]. Analyzing the performance of combined methodologies a lower sensitivity and a progressive higher specificity were evidenced at each examined time. Excluding allograft patients better values of sensitivity, specificity, PV (MCF PPV/NPV:87,5%/83,3% vs RQ-PCR PPV/NPV:100%/75% at T1), LR (MFC LR+/LR−:5,25/0,15 vs RQ-PCR LR+/LR−: +∞/0,25 at T1) were obtained as well as a higher AUC. Combining both methodologies any advantage was achieved. A cut-off value of 10−3 in MFC and 90 wt1-RNA × 104 ABL copies was selected as most significant in terms of risk of relapse and survival (DFS and OS). The median OS of the whole sample was 20,37 months while the DFS was 14,03. As far as DFS is concerned, 57,1% of patients with positive MRD, according to MCF analysis, relapsed with a relapse median time of 11,50 months compared to 22,2% of patients with negative MRD with relapse median time of 29,24 months (p< 0.05).On the other hand, 58,3% patients of high MRD, according to the wt1 expression, relapsed after a median time of 10,97 months compared to 27,3% patients with negative MRD who relapsed after a median time of 29,24 months (p<0,05). Conclusion According to this study, MFC and wt1-RNA copies number are comparable methodologies to detect MRD and to predict relapse. Combination of both does not provide any improvement in the result analysis. At the time of induction therapy we observed the best performance by both methodologies in detecting MRD. In AML MRD levels ≥10−3 in MFC as well as MRD levels ≥ 90 wt1-RNA copies in RQ-PCR, identify two risk groups of patients with different prognosis. Disclosures: No relevant conflicts of interest to declare.

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Vostrikova, Natalya, Daniil Khvostov, Anatoly Zherdev, Mikhail Minaev, and Elena Zvereva. "Development of a two-level control system for the analysis of the composition of meat products." Potravinarstvo Slovak Journal of Food Sciences 15 (October 28, 2021): 1005–17. http://dx.doi.org/10.5219/1632.

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Because of the increased demand for processed meat, there is an urgent need to introduce specific identification methods. Strategies such as molecular genetics and the physical condition of meat are used to quickly explore multi-component products. However, a single methodology does not always unambiguously classify a product as counterfeit. In laboratory practice, as a rule, screening techniques are rarely used in the first stage, followed by arbitration. This work aimed to study individual methodologies using artificially falsified meat samples as examples and to identify their composition based on muscle tissue. For the experiments, the three most common types of raw meat were selected: pork, beef, and chicken. The calculation of the content of muscle tissue was carried out according to the BEFFE method. The study of muscle protein was carried out by ICA, ELISA, PCR, microstructural analysis, and mass spectrometric identification. In this connection, we proposed a multilevel control system for multicomponent meat products. Both classical methodologies, such as calculation by prescription bookmarks (BEFFE) and microstructural analysis, and approaches of highly sensitive methodologies, such as identification of muscle tissue by marker peptides (LC/MS-MRM) and semi-quantitative PCR analysis, were evaluated.

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Santaclara, Francisco J., Montserrat Espiñeira, and Juan M. Vieites. "Genetic Identification of Squids (Families Ommastrephidae and Loliginidae) by PCR–RFLP and FINS Methodologies." Journal of Agricultural and Food Chemistry 55, no. 24 (November 2007): 9913–20. http://dx.doi.org/10.1021/jf0707177.

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Gachet, E., G. G. Martin, F. Vigneau, and G. Meyer. "Detection of genetically modified organisms (GMOs) by PCR: a brief review of methodologies available." Trends in Food Science & Technology 9, no. 11-12 (November 1998): 380–88. http://dx.doi.org/10.1016/s0924-2244(99)00002-3.

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Harvey, M., and F. C. Botha. "Use of PCR-based methodologies for the determination of DNA diversity between Saccharum varieties." Euphytica 89, no. 2 (1996): 257–65. http://dx.doi.org/10.1007/bf00034614.

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Tzanikou, Eleni, Verena Haselmann, Athina Markou, Angelika Duda, Jochen Utikal, Michael Neumaier, and Evi S. Lianidou. "Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients." Clinical Chemistry and Laboratory Medicine (CCLM) 58, no. 11 (October 25, 2020): 1799–807. http://dx.doi.org/10.1515/cclm-2019-0783.

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AbstractBackgroundIn metastatic melanoma, 40%–50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients.MethodsCell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis.ResultsBRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors.ConclusionsOur direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.

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Gaipa, Giuseppe, Giovanni Cazzaniga, Renate E. Panzer-Grümayer, Marinella Veltroni, Leonid Karawajew, Daniela Silvestri, Barbara Buldini, et al. "Time Point-Dependent Concordance of Flow Cytometry and RQ-PCR in the MRD Detection in Childhood ALL: The Experience of the AIEOP-BFM- ALL MRD Study Group." Blood 112, no. 11 (November 16, 2008): 700. http://dx.doi.org/10.1182/blood.v112.11.700.700.

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Abstract In the AIEOP-BFM ALL2000 trial, childhood acute lymphoblastic leukemia (ALL) patients were stratified mainly according to minimal residual disease (MRD) levels at day 33 (Time Point 1, TP1) and 78 (TP2) of treatment, as detected by PCR amplification of clonotypic immunoglobulin and T-cell receptor gene rearrangements. Overall, the results confirmed that PCR-based MRD detection is an independent prognostic indicator which overrides classical risk factors. In this context, MRD measurement by flow cytometry (FCM) on day 15 has been evaluated as an earlier predictor of relapse-free outcome in children with ALL. To understand how these two methods could be applied in a MRD-based clinical protocol, we analyzed the correlation of methodologies in data sets in which PCR and FCM were simultaneously applied. We studied 3,618 BM samples from 1,570 patients (32.5% of all patients on trial) derived from day15 (n=478, 30.4%), 33 (n=1,570) and 78 (n=1,570) of remission induction therapy of the AIEOP-BFM ALL 2000 protocol. Patients were enrolled from September 2000 to June 2006 in Italy, Austria and Germany, and selection was based only on available samples for both PCR and FCM. As in most MRD-based protocols, RQ-PCR was performed on BM mononuclear cells after Ficoll gradient separation, while 4-colors FCM was done on 300,000 nucleated cells (NC) from the whole BM sample after red blood cells lyse-wash procedure. MRD levels ≥10−4 were considered as ‘positive’, while levels below this threshold, not-quantifiable or undetectable were classified as ‘negative’. Overall, qualitative concordance was observed in 2,704/3,618 samples (74.7%) measured at d15, d33 and d78. Concordances at each TP are indicated in the Table. Figure Figure Around 70% of discordant samples in all TPs were cases with low-positivity by PCR (10−4 log range) and negative by FCM, or viceversa. Concordance was also evaluated according to PCR-based MRD risk subgroups. Of note, 519/571 (90.9%) of Standard Risk by PCR-MRD (negative at d33 by 2 markers with sensitivity ≥10−4) were also FCM negative at d33; whereas 59/121 (48.8%) of cases PCR ≥10−3 at d78 (High Risk by PCR-MRD) were FCM negative. In order to evaluate the correlation of methodologies more exactly, we investigated similar cell preparations (same MNC divided for PCR and FCM), and we enhanced the resolution of FCM by using 7-colors and analyzing 500,000 MNC. Samples were collected at days 15, 33, 52, and 78 within a single center. Among a total of 266 samples, the concordance increased up to 87%. More specifically, 100 samples (37.6%) resulted undetectable by both methods; 56 samples were FCM-undetectable and PCR-positive <10−4, while only 13 samples were PCR-positive ≥10−4 but undetectable by FCM. In summary, several methodological issues limit the concordance of FCM and PCR as used by the AIEOP-BFM MRD study group. These include differences in the material analyzed by PCR and FCM, as well as in the amount of sample input (number of cells analyzed). The recent implementation of further technical developments in the FCM procedure, are designed to increase the concordance rate. In conclusion, within BFM-based protocols, FCM cannot simply substitute the current PCR-based MRD risk stratification at the same TPs. Instead, a tailored FCM-based risk definition may be independently reliable. Moreover, the two methodologies applied at different TPs might be complementary in more advanced MRD-based patient stratification protocols. Overall, the choice of the MRD methodology largely depends on the aims of the study protocol, resources available, and treatment strategy.

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Quinteiro, J., C. G. Sotelo, H. Rehbein, S. E. Pryde, I. Medina, R. I. Pérez-Martín, M. Rey-Méndez, and I. M. Mackie. "Use of mtDNA Direct Polymerase Chain Reaction (PCR) Sequencing and PCR−Restriction Fragment Length Polymorphism Methodologies in Species Identification of Canned Tuna." Journal of Agricultural and Food Chemistry 46, no. 4 (April 1998): 1662–69. http://dx.doi.org/10.1021/jf970552+.

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Santos, Carlos Ferreira dos, Vivien Thiemy Sakai, Maria Aparecida de Andrade Moreira Machado, Daniela Nicole Schippers, and Andrew Seth Greene. "Reverse transcription and polymerase chain reaction: principles and applications in dentistry." Journal of Applied Oral Science 12, no. 1 (March 2004): 1–11. http://dx.doi.org/10.1590/s1678-77572004000100002.

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Various molecular biology techniques have become available in the last few years. One of the most revolutionary of these techniques regarding nucleic acid analysis is the polymerase chain reaction (PCR), which was first described in 1985. This method relies on the exponential amplification of specific DNA fragments, resulting in millions of copies that can serve as templates for different kinds of analyses. PCR can be preceded by a reverse transcription (RT) reaction in order to produce cDNA from RNA (RT-PCR). RT-PCR provides the possibility to assess gene transcription in cells or tissues. PCR and RT-PCR techniques have been instrumental in dental research, and show potential to be used for diagnosis as well as for treatment and prevention of many diseases (dental caries, periodontal disease, endodontic infections and oral cancer). Compared to other traditional methodologies, PCR and RT-PCR show many advantages including high specificity, sensitivity, and speed. Since PCR and RT-PCR are relatively new techniques and are not available to most students and professionals involved with dentistry, the aim of this work is to present the details of these techniques as well as dental literature reports in which they were used.

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Busque, Lambert, Yves Paquette, Sylvie Provost, Denis-Claude Roy, Ross L. Levine, Luigina Mollica, and D. Gary Gilliland. "Skewing of X-inactivation ratios in blood cells of aging women is confirmed by independent methodologies." Blood 113, no. 15 (April 9, 2009): 3472–74. http://dx.doi.org/10.1182/blood-2008-12-195677.

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Abstract Nonrandom X-chromosome inactivation (XCI), also known as skewing, has been documented in the blood cells of a significant proportion of normal aging women by the use of methylation-based assays at the polymorphic human androgen receptor locus (HUMARA). Recent data obtained with a new transcription-based XCI determination method, termed suppressive polymerase chain reaction (PCR), has shed controversy over the validity of XCI ratio results obtained with HUMARA. To resolve this disparity, we analyzed XCI in polymorphonuclear leukocytes of a large cohort of women aged 43 to 100 years with the use of HUMARA (n = 100), a TaqMan single nucleotide polymorphism (SNP) assay (n = 90), and the suppressive polymerase chain reaction (PCR) assay (n = 67). The 3 methods yielded similar skewing incidences (42%, 38%, and 40%, respectively), and highly concordant XCI ratios. This confirms that the skewing of XCI ratio seen in blood cells of aging women is a bona fide and robust biologic phenomenon.

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Patton, Janet C., Eveline Akkers, Ashraf H. Coovadia, Tammy M. Meyers, Wendy S. Stevens, and Gayle G. Sherman. "Evaluation of Dried Whole Blood Spots Obtained by Heel or Finger Stick as an Alternative to Venous Blood for Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Vertically Exposed Infants in the Routine Diagnostic Laboratory." Clinical and Vaccine Immunology 14, no. 2 (December 13, 2006): 201–3. http://dx.doi.org/10.1128/cvi.00223-06.

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ABSTRACT The diagnostic accuracy of the Roche Amplicor human immunodeficiency virus type 1 DNA PCR assay (version 1.5) on DNA extracted from pediatric heel prick dried blood spots using Roche MagNA Pure nucleic acid purification technology was evaluated. The methodologies transfer successfully from the labor-intensive research laboratory to the high-throughput automated routine laboratory.

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Wallander, Michelle L., Katherine B. Geiersbach, Sheryl R. Tripp, and Lester J. Layfield. "Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing." Archives of Pathology & Laboratory Medicine 136, no. 7 (July 1, 2012): 796–803. http://dx.doi.org/10.5858/arpa.2011-0321-oa.

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Context.—Echinoderm microtubule–associated proteinlike 4–anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non–small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. Objective.—To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Design.—Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n = 42; mutant, n = 4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). Results.—EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. Conclusions.—The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

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Settanni, Luca, Douwe van Sinderen, Jone Rossi, and Aldo Corsetti. "Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR." Applied and Environmental Microbiology 71, no. 6 (June 2005): 3049–59. http://dx.doi.org/10.1128/aem.71.6.3049-3059.2005.

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ABSTRACT A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.

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Perrey, Chris, Stephen J. Turner, Vera Pravica, W. Martin Howell, and Ian V. Hutchinson. "ARMS-PCR methodologies to determine IL-10, TNF-α, TNF-β and TGF-β1 gene polymorphisms." Transplant Immunology 7, no. 2 (April 1999): 127–28. http://dx.doi.org/10.1016/s0966-3274(99)80030-6.

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Fajardo, Violeta, Isabel González, María Rojas, Teresa García, and Rosario Martín. "A review of current PCR-based methodologies for the authentication of meats from game animal species." Trends in Food Science & Technology 21, no. 8 (August 2010): 408–21. http://dx.doi.org/10.1016/j.tifs.2010.06.002.

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Jerez Puebla, Luis Enrique, Fidel A. Núñez Fernández, Jorge Fraga, Lázara Rojas Rivero, Iraís Atencio Millán, Lucía Ayllón Valdés, Isabel Martínez Silva, Norbert Müller, and Lucy J. Robertson. "Concordance of Giardia duodenalis assemblages determined by different PCR methodologies in three observational studies in Cuba." Experimental Parasitology 209 (February 2020): 107814. http://dx.doi.org/10.1016/j.exppara.2019.107814.

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Herrero, Beatriz, María Madriñán, Juan M. Vieites, and Montserrat Espiñeira. "Rapid Identification of Seaweeds in Food Products by PCR Combined with ALF-RFLP and FINS Methodologies." Journal of Agricultural and Food Chemistry 58, no. 22 (November 24, 2010): 11586–92. http://dx.doi.org/10.1021/jf103464b.

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Houhoula, Dimitra Panagiotis, Vasilios Belsis, Leonidas Georgopoulos, Virginia Giannou, Vasiliki R. Kyrana, John Tsaknis, Vladimiros P. Lougovois, and Stamatios Koussissis. "Detection of Sesame Allergen Traces with Two PCR Assays - The Challenge to Protect Food-Allergic Consumers." Turkish Journal of Agriculture - Food Science and Technology 3, no. 4 (January 13, 2015): 210. http://dx.doi.org/10.24925/turjaf.v3i4.210-215.221.

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The purpose of this study was to investigate the possible presence of sesame in commercial foods normally carrying no warning for the allergen, but which may have been subjected to contamination during processing. One hundred units of widely consumed goods with high potential to contain allergenic substances deriving from nuts were analyzed, using sensitive and capable PCR (C-PCR) and Real Time PCR (RT-PCR) methodologies. Of the products examined, 15 (15.0%) declared the presence of sesame, 36 (36.0%) carried no food allergy label, 44 (44.0%) were marked by the phrase “may contain traces of nuts” and 5 (5.0%) carried the indication “may contain sesame traces”. The sesame-positive products detected using the C-PCR method were 15 (100%), 12 (33.3%), 14 (31.8%) and 3 (60%), respectively. Using the RT-PCR technique, positive results were obtained for 15 (100%), 18 (50.0%), 18 (20.5%) and 5 (100%) samples, respectively. The results indicate that the PCR methods applied are highly sensitive and selective, which makes them suitable for the detection of sesame traces in food samples. In addition, they can be useful for monitoring the effectiveness of cleaning processes in the production units of the food industry.

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Petiti, Jessica, Valentina Rosso, Eleonora Croce, Vanessa Franceschi, Giacomo Andreani, Matteo Dragani, Marco De Gobbi, et al. "Highly Sensitive Detection of IDH2 Mutations in Acute Myeloid Leukemia." Journal of Clinical Medicine 9, no. 1 (January 19, 2020): 271. http://dx.doi.org/10.3390/jcm9010271.

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Background: Acute myeloid leukemia is a heterogeneous hematological disease, characterized by karyotypic and molecular alterations. Mutations in IDH2 have a role in diagnosis and as a minimal residue disease marker. Often the variant allele frequency during follow up is less than 20%, which represents the limit of detection of Sanger sequencing. Therefore, the development of sensitive methodologies to identify IDH2 mutations might help to monitor patients’ response to therapy. We compared three different methods to identify and monitor IDH2 mutations in patients’ specimens. Methods: Performances of PNA-PCR clamping, droplet digital PCR and Sanger for IDH2 status identification were evaluated and compared in 96 DNA patients’ specimens. Results: In contrast with Sanger sequencing, our results highlighted the concordance between PNA clamping and digital PCR. Furthermore, PNA-PCR clamping was able to detect more mutated DNA with respect to Sanger sequencing that showed several false negatives independently from the allelic frequency. Conclusions: We found that PNA-PCR clamping and digital PCR identified IDH2 mutations in DNA samples with comparable results in a percentage significantly higher compared to Sanger sequencing. PNA-PCR clamping can be used even in laboratories not equipped for sophisticated analyses, decreasing cost and time for IDH2 characterization.

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Siggillino, Annamaria, Paola Ulivi, Luigi Pasini, Maria Sole Reda, Elisa Chiadini, Francesca Romana Tofanetti, Sara Baglivo, et al. "Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR." Diagnostics 10, no. 12 (December 7, 2020): 1062. http://dx.doi.org/10.3390/diagnostics10121062.

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Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

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Neagu, Monica, Carolina Constantin, and Mihaela Surcel. "Testing Antigens, Antibodies, and Immune Cells in COVID-19 as a Public Health Topic—Experience and Outlines." International Journal of Environmental Research and Public Health 18, no. 24 (December 14, 2021): 13173. http://dx.doi.org/10.3390/ijerph182413173.

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The current COVID-19 pandemic has triggered an accelerated pace in all research domains, including reliable diagnostics methodology. Molecular diagnostics of the virus and its presence in biological samples relies on the RT-PCR method, the most used and validated worldwide. Nonconventional tests with improved parameters that are in the development stages will be presented, such as droplet digital PCR or CRISPR-based assays. These molecular tests were followed by rapid antigen testing along with the development of antibody tests, whether based on ELISA platform or on a chemiluminescent microparticle immunoassay. Less-conventional methods of testing antibodies (e.g., lateral flow immunoassay) are presented as well. Left somewhere in the backstage of COVID-19 research, immune cells and, furthermore, immune memory cells, are gaining the spotlight, more so in the vaccination context. Recently, methodologies using flow-cytometry evaluate circulating immune cells in infected/recovered patients. The appearance of new virus variants has triggered a surge for tests improvement. As the pandemic has entered an ongoing or postvaccination era, all methodologies that are used to monitor public health focus on diagnostic strategies and this review points out where gaps should be filled in both clinical and research settings.

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Lawson, A. J., J. M. J. Logan, G. L. O'neill, M. Desai, and J. Stanley. "Large-Scale Survey of CampylobacterSpecies in Human Gastroenteritis by PCR and PCR–Enzyme-Linked Immunosorbent Assay." Journal of Clinical Microbiology 37, no. 12 (1999): 3860–64. http://dx.doi.org/10.1128/jcm.37.12.3860-3864.1999.

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A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured “Campylobacterspp.” from 464 samples. The PCR methodologies detected 492Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli(PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections withC. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. larisuggests that it is less important in this context.

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Haworth, R., and A. M. Pilling. "The PCR assay in the preclinical safety evaluation of nucleic acid medicines." Human & Experimental Toxicology 19, no. 5 (May 2000): 267–76. http://dx.doi.org/10.1191/096032700678815891.

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The polymerase chain reaction (PCR) is a highly efficient gene amplification procedure which is increasingly being applied to the safety assessment of nucleic acid (NA) medicines such as gene therapies and DNA vaccines. Although clinical experience is limited, a number of potential safety issues exist with these new compounds including toxicity associated with the expression of encoded gene products, autoimmunity due to the induction of anti-DNA antibodies and insertional mutagenesis. PCR enables these questions to be addressed and provides data on mRNA expression, biodistribution and integration. In this review the use of PCR methodologies in the preclinical safety evaluation of NA medicines is discussed. Particular consideration is given to the issues surrounding the use of PCR in regulatory toxicology, including sensitiv-ity requirements, cross-contamination problems, tissue sampling procedures and good laboratory practice (GLP) compliance. In addition, the use of a PCR-based assay to demonstrate the integration of DNA vector into host DNA is described. As the use of PCR in the development of NA medicines will undoubtedly increase over the next few years, it is important that pathologists and toxicologists familiarise themselves with the principles and applications of this technique.

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Williamson, Helen, Edward Mountjoy, Hashem Shihab, Jack Bartram, Mike Hubank, Nicholas Goulden, Ian Day, Eileen Roberts, John Moppett, and Jeremy Hancock. "Development Of Minimal Residual Disease (MRD) Analysis In Childhood Acute Lymphoblastic Leukaemia (ALL) By Next Generation Sequencing (NGS)." Blood 122, no. 21 (November 15, 2013): 2569. http://dx.doi.org/10.1182/blood.v122.21.2569.2569.

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Abstract Detection of sub-microscopic levels of disease (minimal residual disease; MRD) in childhood acute lymphoblastic leukaemia (ALL) during treatment is an important prognostic factor. Currently, stratification of therapy for the new frontline trial in childhood ALL (UKALL 2011) is provided by MRD analysis using real time quantitative PCR (RQ-PCR) to identify and quantitate the patient specific rearrangements of the immunoglobulin (Ig) and T-cell receptor (TCR) genes. The current methodology is expensive, time-consuming and complex to perform. Although MRD has proven to be a powerful and essential tool in stratification of ALL patients, 8% of individuals in the current UKALL 2011 trial do not have an informative MRD result. Recently, Next Generation Sequencing (NGS) has led to the opportunity to improve the sensitivity and specificity of Ig/TCR based MRD analysis. In this study, we focussed on the IgH locus using BIOMED 2 primers (van Dongen et al., 2003) modified to allow target identification and quantitation by deep sequencing on the Illumina MiSeq platform. We developed a novel pipeline to automate the clustering and classification of sequencing reads leading to characterisation of the clonal subtypes present. In a sample of 12 patients, the method correctly identified all the major clones revealed by current methodologies, and also detected many related and unrelated low-frequency clones. Additional targets were also identified in patients in which no IgH targets were detectable by current methodologies. These NGS-identified targets were subsequently used to monitor MRD by RQ-PCR to the desired quantitative range required for stratification of therapy according to UKALL 2011 guidelines (Figure 1). In addition, we were able to delineate patterns of IgH rearrangements in two patients previously shown to have oligoclonal (>2) rearrangements. Such patients represent a time consuming and technical challenge for current technologies as it is important that all targets at the locus are followed by RQ-PCR to provide an informative and robust MRD result. Furthermore, by clustering similar sequences, we identified diagnostic samples where multiple V regions are attached to the same N1-D-N2-J region. This may allow for the study of clonal evolution in follow-up samples. Altogether, NGS sequencing has the potential to significantly reduce false negative results, as multiple evolved clones can be identified. This methodology also represents a significant time saving (5-7 days) in comparison to established methods (3-4 weeks).Figure 1.(a) Polyacrylamide electrophoresis could not recognise a target to use in current MRD methodologies (well 1 containing the products from a PCR reaction that would amplify VH1 and VH7, and wells 2-6 amplifying VH2-6, respectively), while the NGS pipeline could identify a VH7 rearrangement (b). (c) ASOs were designed to amplify the NGS-identified VH7-81*01 DH3-9*01 JH4*02 rearrangement and optimised to correctly identify 10-2, 10-3, 10-4 dilutions with a single NAC (non-amplification control; monocytes from 20 normal individuals) replicate amplified, therefore meeting current guidelines for a MRD target.Figure 1. (a) Polyacrylamide electrophoresis could not recognise a target to use in current MRD methodologies (well 1 containing the products from a PCR reaction that would amplify VH1 and VH7, and wells 2-6 amplifying VH2-6, respectively), while the NGS pipeline could identify a VH7 rearrangement (b). (c) ASOs were designed to amplify the NGS-identified VH7-81*01 DH3-9*01 JH4*02 rearrangement and optimised to correctly identify 10-2, 10-3, 10-4 dilutions with a single NAC (non-amplification control; monocytes from 20 normal individuals) replicate amplified, therefore meeting current guidelines for a MRD target. Having established NGS for identifying clonal targets in ALL, we are currently assessing the ability of the method and pipeline to quantify disease levels in end of induction and relapse samples, previously analysed by RQ-PCR, to determine the concordance between the methodologies. Indeed, logarithmic dilution series of patient DNA in a normal background revealed that stratification based on a clinical threshold of 1 in 1,000,000 is possible using this methodology. Further investigation into the clinical utility of NGS for MRD analysis will focus on analysing earlier time points in treatment and studying the potential use of blood rather than bone marrow. Altogether, this will further improve the predictive value and specificity of MRD testing. Disclosures: No relevant conflicts of interest to declare.

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Roellig, Dawn M., Luis A. Gomez-Puerta, Daniel G. Mead, Jesus Pinto, Jenny Ancca-Juarez, Maritza Calderon, Caryn Bern, Robert H. Gilman, and Vitaliano A. Cama. "Hemi-Nested PCR and RFLP Methodologies for Identifying Blood Meals of the Chagas Disease Vector, Triatoma infestans." PLoS ONE 8, no. 9 (September 11, 2013): e74713. http://dx.doi.org/10.1371/journal.pone.0074713.

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Scollo, Francesco, Leticia A. Egea, Alessandra Gentile, Stefano La Malfa, Gabriel Dorado, and Pilar Hernandez. "Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies." Food Chemistry 213 (December 2016): 388–94. http://dx.doi.org/10.1016/j.foodchem.2016.06.086.

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Agriculture, G. Douglas Inglis, Matthew C. Thomas, Dallas K. Thomas, Martin L. Kalmokoff, Stephen P. J. Brooks, and L. Brent Selinger. "Molecular Methods to Measure Intestinal Bacteria: A Review." Journal of AOAC INTERNATIONAL 95, no. 1 (January 1, 2012): 5–23. http://dx.doi.org/10.5740/jaoacint.sge_inglis.

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Abstract The intestine is an exceptionally rich ecosystem encompassing a complex interaction among microorganisms, influenced by host factors, ingested food, and liquid. Characterizing the intestinal microbiota is currently an active area of research. Various molecular-based methods are available to characterize the intestinal microbiota, but all methods possess relative strengths, as well as salient weaknesses. It is important that researchers are cognizant of the limitations of these methods, and that they take the appropriate steps to mitigate weaknesses. Here, we discuss methodologies used to monitor intestinal bacteria including: (i) traditional clone libraries; (ii) direct sequencing using next-generation parallel sequencing technology; (iii) denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis; (iv) terminal restriction fragment length polymorphism analysis; (v) fluorescent in situ hybridization; and (vi) quantitative PCR. In addition, we also discuss experimental design, sample collection and storage, DNA extraction, gene targets, PCR bias, and methods to reduce PCR bias.

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Morehouse, Zachary P., Lyson Samikwa, Caleb M. Proctor, Harry Meleke, Mercy Kamdolozi, Gabriella L. Ryan, David Chaima, Antonia Ho, Rodney J. Nash, and Tonney S. Nyirenda. "Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: A model for reducing resources needed for accurate testing." PLOS ONE 16, no. 8 (August 18, 2021): e0256316. http://dx.doi.org/10.1371/journal.pone.0256316.

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Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.

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Dahal, Pragyan, Basudha Khanal, Keshav Rai, Vivek Kattel, Satish Yadav, and Narayan Raj Bhattarai. "Challenges in Laboratory Diagnosis of Malaria in a Low-Resource Country at Tertiary Care in Eastern Nepal: A Comparative Study of Conventional vs. Molecular Methodologies." Journal of Tropical Medicine 2021 (December 28, 2021): 1–9. http://dx.doi.org/10.1155/2021/3811318.

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For ongoing malaria elimination programmes, available methods such as microscopy and rapid diagnostic tests (RDTs) cannot detect all malaria cases in acute febrile illness. These methods are entirely dependent on the course of infection, parasite load, and skilled technical resources. Our study objectives were to estimate the performance of light microscopy and a RDT as well as real-time PCR for the detection of the Plasmodium parasite. Altogether, 52 blood samples collected from patients with acute febrile illness were tested by microscopy, RDT, and real-time PCR. The results were compared in terms of sensitivity and specificity. Microscopy detected the malaria parasite in 5.8% of the blood samples whereas 13.5% were detected by the RDT and 27% by real-time PCR. Considering real-time PCR as the gold standard method, microscopy had a sensitivity of 21.4% and a specificity of 100%, and the RDT had a sensitivity of 28.6% and a specificity of 92.1%. Microscopy together with the RDT successfully detected malaria positive cases in blood samples of Ct value below 20, but both were unable to detect malaria cases between 26–40 Ct value ranges amplified by real-time PCR. Despite various diagnostic tools being available, microscopy still remains the method of choice for diagnosis, while the RDT is user-friendly when applied at the point of care. However, our preliminary results emphasize the need to implement the test with higher sensitivity and specificity in the context of a malaria elimination programme. Such programmes can be a crucial opportunity to understand the species prevalent in a low-endemic region. However, these results should be further verified with a large cohort study to document the submicroscopic infection.

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Kenten, J. H., J. Casadei, J. Link, S. Lupold, J. Willey, M. Powell, A. Rees, and R. Massey. "Rapid electrochemiluminescence assays of polymerase chain reaction products." Clinical Chemistry 37, no. 9 (September 1, 1991): 1626–32. http://dx.doi.org/10.1093/clinchem/37.9.1626.

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Abstract We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

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Chen, Jin-Qiang, Stephanie Healey, Patrick Regan, Pongpan Laksanalamai, and Zonglin Hu. "PCR-based methodologies for detection and characterization of Listeria monocytogenes and Listeria ivanovii in foods and environmental sources." Food Science and Human Wellness 6, no. 2 (June 2017): 39–59. http://dx.doi.org/10.1016/j.fshw.2017.03.001.

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Galal-Khallaf, Asmaa, Khaled Mohammed-Geba, Alaa G. M. Osman, Khaled Y. AbouelFadl, Yaisel J. Borrell, and Eva Garcia-Vazquez. "SNP-based PCR-RFLP, T-RFLP and FINS methodologies for the identification of commercial fish species in Egypt." Fisheries Research 185 (January 2017): 34–42. http://dx.doi.org/10.1016/j.fishres.2016.09.031.

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Cardus, Beatrix, Richard Colling, Angela Hamblin, and Elizabeth Soilleux. "Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens." Journal of Clinical Pathology 72, no. 6 (March 14, 2019): 406–11. http://dx.doi.org/10.1136/jclinpath-2019-205734.

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AimsBRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT).Methods11 HCL, 5 HCL ‘mimic’, 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC).ResultsPCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples.ConclusionsPCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.

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Filliol, Ingrid, Severine Ferdinand, Laetitia Negroni, Christophe Sola, and Nalin Rastogi. "Molecular Typing of Mycobacterium tuberculosis Based on Variable Number of Tandem DNA Repeats Used Alone and in Association with Spoligotyping." Journal of Clinical Microbiology 38, no. 7 (2000): 2520–24. http://dx.doi.org/10.1128/jcm.38.7.2520-2524.2000.

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Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of Mycobacterium tuberculosis in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS6110restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS6110 RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.

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Bakaletz, Lauren O., Gregory J. White, J. Christopher Post, and Garth D. Ehrlich. "Blinded Multiplex PCR Analyses of Middle Ear and Nasopharyngeal Fluids from Chinchilla Models of Single- and Mixed-Pathogen-Induced Otitis Media." Clinical Diagnostic Laboratory Immunology 5, no. 2 (March 1, 1998): 219–24. http://dx.doi.org/10.1128/cdli.5.2.219-224.1998.

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ABSTRACT Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at −70°C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeable H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.

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Pereira, Mariana R., Fabiana Rocha-Silva, Cidiane Graciele-Melo, Camila R. Lafuente, Telcia Magalhães, and Rachel B. Caligiorne. "Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis." BioMed Research International 2014 (2014): 1–4. http://dx.doi.org/10.1155/2014/639310.

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The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome ofLeishmaniaspecies in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12) presented positive results for serology and 79% (n=15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

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Sooai, Christiane Marlene, Elsa Herdiana M, and Supargiyono Supargiyono. "OPTIMAL CONDITION FOR MULTIPLEX POLYMERASE CHAIN REACTION (PCR) IN DETECTING ASCARIS LUMBRICOIDES, TRICHURIS TRICHIURA, AND NECATOR AMERICANUS IN PRESERVED STOOL." Berkala Ilmiah Kedokteran Duta Wacana 5, no. 1 (August 19, 2020): 34. http://dx.doi.org/10.21460/bikdw.v5i1.171.

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Background: Multiplex PCR examination is one of the molecular examination methodologies applied to detect soil-transmitted helminth (STH) infection. Optimization of the multiplex PCR method is a complex process, but it is necessary to obtain both a correct detection process and a satisfactory DNA product. Objective: To determine whether multiplex PCR can be optimized to diagnose STH from Indonesian isolates, and to find the optimal method for detection of A. lumbricoides, T. trichiura, and N. americanus infections in stool that have been stored for 3 years. Methods: A total of 15 samples were examined, and these samples were previously examined by using a microscopic method, then continued with optimization steps. Result: The optimal PCR mixture used primers targeting COI gene for A. lumbricoides, 18S rDNA for T. trichiura and ITS1 for N. americanus, 15 µl of Go Taq Green Master Mix, 5 µl of the 3 pairs of primers, 5 µl of DNA template and 4 µl of DdH2O, and the condition was 30 minutes of 950C denaturation, 30 second of 530C annealing and 1 minute of 720C extension, repeated for 35 cycles. Conclusion: Multiplex PCR can be optimized for STH detection from Indonesian isolates. The successful detection using the multiplex PCR method was influenced by sample preparation prior to DNA isolation, which includes several steps i.e. homogenization of samples using bead beaters and passing samples on liquid nitrogen rapidly.

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Journal articles on the topic 'PCR methodologies'

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