Journal articles: 'Isothermal pcr'

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Park, Jee-Woong. "Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests." Biosensors 12, no. 10 (October 10, 2022): 857. http://dx.doi.org/10.3390/bios12100857.

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For the identification of nucleic acids, which are important biomarkers of pathogen-mediated diseases and viruses, the gold standard for NA-based diagnostic applications is polymerase chain reaction (PCR). However, the requirements of PCR limit its application as a rapid point-of-care diagnostic technique. To address the challenges associated with regular PCR, many isothermal amplification methods have been developed to accurately detect NAs. Isothermal amplification methods enable NA amplification without changes in temperature with simple devices, as well as faster amplification times compared with regular PCR. Of the isothermal amplifications, loop-mediated isothermal amplification (LAMP) is the most studied because it amplifies NAs rapidly and specifically. This review describes the principles of LAMP, the methods used to monitor the process of LAMP, and examples of biosensors that detect the amplicons of LAMP. In addition, current trends in the application of LAMP to smartphones and self-diagnosis systems for point-of-care tests are also discussed.

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Yunita, Lisa, Dian Rachma Wijayanti, and Apriani Riyanti. "Sensitivitas Pemeriksaan Covid-19: Insulated Isothermal PCR (iiPCR) Dan Reverse Transcription PCR (RT-PCR)." JOURNAL OF MUHAMMADIYAH MEDICAL LABORATORY TECHNOLOGIST 6, no. 1 (May 19, 2023): 37. http://dx.doi.org/10.30651/jmlt.v6i1.12441.

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Covid-19 (Corona Virus disease 2019) is a new type of disease caused by a virus from the Corona Virus group, namely SARS-CoV-2. Covid-19 may cause respiratory system disorders, ranging from mild symptoms such as flu to lung infections, such as pneumonia. Laboratory diagnoses for Covid-19 disease generally include hematological (complete blood examinations), and molecular or a combination of serology and molecular. PCR examination that can be carried out using the Insulated Isothermal PCR and Reverse Transcription PCR methods. This research is descriptive observational research. Data were collected at Grha Kedoya Hospital, West Jakarta, North Kedoya. The result showed a sensitivity of iiPCR score 82,6%, which is relatively lower than gold-standard RT-PCR. Our research suggested that RT-PCR is still an effective and sensitive method for Covid-19 examination.

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Bouam, Amar, Jean-Jacques Vincent, Elisabeth Le Glass, Lionel Almeras, Pierre-Yves Levy, Hervé Tissot-Dupont, Jean-Christophe Lagier, Pierre-Edward Fournier, Didier Raoult, and Michel Drancourt. "Rapid Isothermal Amplification for the Buccal Detection SARS-CoV-2 in the Context of Out-Patient COVID-19 Screening." Journal of Clinical Medicine 10, no. 12 (June 16, 2021): 2643. http://dx.doi.org/10.3390/jcm10122643.

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A commercially available isothermal amplification of SARS-CoV-2 RNA was applied to self-collected saliva samples using dry dental cotton rolls, which were held in the mouth for two minutes. Of 212 tests, isothermal amplification yielded three (0.14%) invalid results, 120 (56.6%) positive results and 89 (42%) negative results. Compared to reference RT-PCR assays routinely performed simultaneously on nasopharyngeal swabs, excluding the three invalid isothermal amplification assays and one RT-PCR invalid assay, these figures indicated that 119/123 (96.7%) samples were positive in both methods and 85/85 samples were negative in both methods. Four positive buccal swabs which were missed by the isothermal amplification, exhibited Ct values of 26–34 in reference RT-PCR assays. Positive isothermal amplification detection was achieved in less than 10 min. Supervision of the self-sampling procedure was key to achieve these performances. These data support the proposal to use the protocol reported in this paper, including supervised buccal self-sampling, to screen people suspected of having COVID-19 at the point of care.

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Shahrajabian, Mohamad H., Wenli Sun, and Qi Cheng. "Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus." Current Pharmaceutical Design 27, no. 25 (September 15, 2021): 2893–903. http://dx.doi.org/10.2174/1381612827666210604114411.

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Introduction: While PCR has been recognized as one of the appropriate ways to diagnose infectious diseases, Loop-mediated isothermal amplification (LAMP) which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords included PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA involves benefits of isothermal PCR as well as simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and involves a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.

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Ahrberg, Christian D., Andreas Manz, and Bong Geun Chung. "Polymerase chain reaction in microfluidic devices." Lab on a Chip 16, no. 20 (2016): 3866–84. http://dx.doi.org/10.1039/c6lc00984k.

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Maltzeva, Yulia I., Daria A. Gorbenko, Ekaterina V. Nikitina, Maria S. Rubel, and Dmitry M. Kolpashchikov. "Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM)." International Journal of Molecular Sciences 24, no. 9 (April 25, 2023): 7812. http://dx.doi.org/10.3390/ijms24097812.

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Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population’s health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

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Rostamkhani, N., A. Haghnazari, M. Tohidfar, and A. Moradi. "Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification." Czech Journal of Genetics and Plant Breeding 47, No. 4 (December 15, 2011): 140–48. http://dx.doi.org/10.17221/7/2011-cjgpb.

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In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T2 transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65°C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10–1 to 10–8) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

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TSEN, HAU-YANG, CHIA-MING SHIH, PING-HUA TENG, HSIN-YEN CHEN, CHIA-WEI LIN, CHIEN-SHUN CHIOU, HWA-TANG THOMAS WANG, et al. "Detection of Salmonella in Chicken Meat by Insulated Isothermal PCR." Journal of Food Protection 76, no. 8 (August 1, 2013): 1322–29. http://dx.doi.org/10.4315/0362-028x.jfp-12-553.

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Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5′ and 3′ ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 100 CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

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Du, Tian, Ji-hong Lin, Jun-hua Zhao, Hai-bo Wang, and Qiu-hua Mo. "Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System." Canadian Journal of Infectious Diseases and Medical Microbiology 2020 (September 7, 2020): 1–5. http://dx.doi.org/10.1155/2020/9373984.

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Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

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Daher, Rana K., Gale Stewart, Maurice Boissinot, and Michel G. Bergeron. "Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples." Clinical Chemistry 60, no. 4 (April 1, 2014): 660–66. http://dx.doi.org/10.1373/clinchem.2013.213504.

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Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.

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Chen, Jyh Jian, Wei Hua Chen, and Yi Shiang Shie. "The Effect of Thermal Contact Resistance on Heat Management in a Shuttling PCR System." Applied Mechanics and Materials 284-287 (January 2013): 1941–45. http://dx.doi.org/10.4028/www.scientific.net/amm.284-287.1941.

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A novel shuttling polymerase chain reaction (PCR) system is assembled to make temperature uniform in the reaction chamber. The chamber is oscillated by a servo motor and contacted with three different isothermal zones to complete several thermal cycles. The home-made computer code is utilized to investigate the influences of operational parameters on the temperature inside the chamber. Numerical results show that the contact resistances between the heating blocks and the reaction chamber dominate the temperatures inside the PCR chamber. In this work a PCR system that is composed of the PID controller, the moving stage, three aluminum blocks for three different isothermal zones and a reaction chamber is also developed. Experimental results demonstrated that the stability of this shuttling PCR system is confirmed. And results show that DNA templates provided with the yT&A® cloning vector are amplified successfully in this PCR system.

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Reif, John H., and Urmi Majumder. "Isothermal reactivating Whiplash PCR for locally programmable molecular computation." Natural Computing 9, no. 1 (August 9, 2009): 183–206. http://dx.doi.org/10.1007/s11047-009-9148-6.

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Maruyama, Fumito, Takehiko Kenzaka, Nobuyasu Yamaguchi, Katsuji Tani, and Masao Nasu. "Detection of Bacteria Carrying the stx2 Gene by In Situ Loop-Mediated Isothermal Amplification." Applied and Environmental Microbiology 69, no. 8 (August 2003): 5023–28. http://dx.doi.org/10.1128/aem.69.8.5023-5028.2003.

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ABSTRACT A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA 2 in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA 2 gene. Higher-contrast images were obtained with this method than with in situ PCR.

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James, Ameh, and John Alawneh. "COVID-19 Infection Diagnosis: Potential Impact of Isothermal Amplification Technology to Reduce Community Transmission of SARS-CoV-2." Diagnostics 10, no. 6 (June 11, 2020): 399. http://dx.doi.org/10.3390/diagnostics10060399.

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The current coronavirus disease 2019 (COVID-19) pandemic is largely driven by community transmission, after 2019 novel Coronavirus (2019-nCoV or SARS-CoV-2) crosses the borders. To stop the spread, rapid testing is required at community clinics and hospitals. These rapid tests should be comparable with the standard PCR technology. Isothermal amplification technology provides an excellent alternative that is highly amenable to resource limited settings, where expertise and infrastructure to support PCR are not available. In this review, we provide a brief description of isothermal amplification technology, its potential and the gaps that need to be considered for SARS-CoV-2 detection. Among this emerging technology, loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA) and Nicking enzyme-assisted reaction (NEAR) technologies have been identified as potential platforms that could be implemented at community level, without samples referral to a centralized laboratory and prolonged turnaround time associated with the standard COVID-19 RT-PCR test. LAMP, for example, has recently been shown to be comparable with PCR and could be performed in less than 30 min by non-laboratory staff, without RNA extractions commonly associated with PCR. Interestingly, NEAR (ID NOW™ COVID-19 (Abbott, IL, USA) was able to detect the virus in 5 min. More so, isothermal platforms are cost effective and could easily be scaled up to resource limited settings. Diagnostics developers, scientific community and commercial companies could consider this alternative method to help stop the spread of COVID-19.

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García-Bernalt Diego, Juan, Pedro Fernández-Soto, Begoña Febrer-Sendra, Beatriz Crego-Vicente, and Antonio Muro. "Loop-Mediated Isothermal Amplification in Schistosomiasis." Journal of Clinical Medicine 10, no. 3 (February 1, 2021): 511. http://dx.doi.org/10.3390/jcm10030511.

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Human schistosomiasis is one of the most important parasitic diseases, causing around 250 million cases (mostly in Africa) and 280,000–500,000 deaths every year. Due to the limited resources and the far-removed nature of many endemic areas, the implementation of new, sensitive and specific diagnostic tools has had little success. This is particularly true for PCR-based molecular methods that require expensive equipment and trained personnel to be executed. Loop-mediated isothermal amplification (LAMP) along with other isothermal techniques appeared in the early 21st century as an alternative to those methods, overcoming some of the aforementioned limitations and achieving a more inexpensive diagnostic. However, to this date, neither LAMP nor any other isothermal technique have signified a meaningful change in the way schistosomiasis diagnosis is routinely performed. Here, we present the recent developments in LAMP-based schistosomiasis diagnosis. We expose the main advantages and disadvantages of LAMP technology over PCR and other classical diagnostic methods focusing in various research approaches on intermediate hosts, animal models and patients. We also examine its potential clinical application in post-therapy monitoring, as well as its usefulness as a point-of-care test.

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Eckel, Florian, Franziska Küsters, Bernhard Drossel, Markus Konert, Hans Mattes, and Stefan Schopf. "Variplex™ test system fails to reliably detect SARS-CoV-2 directly from respiratory samples without RNA extraction." European Journal of Clinical Microbiology & Infectious Diseases 39, no. 12 (July 17, 2020): 2373–77. http://dx.doi.org/10.1007/s10096-020-03983-9.

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AbstractDiagnosis of COVID is performed by PCR methods, but their capacity is limited by the requirement of high-level facilities and instruments. The loop-mediated isothermal amplification (LAMP) method has been utilized for the detection of isolated virus-specific RNA. Preliminary data suggest the possibility of isothermal amplification directly from respiratory samples without RNA extraction. All patients admitted to our hospital were screened for SARS-CoV-2 by routine. Respiratory samples were tested by variplex system based on LAMP method directly without RNA extraction and by PCR. Primary endpoint was the false-negative rate of variplex test compared with PCR as gold standard. In 109 patients variplex test and PCR assay were performed simultaneously. Median age was 80 years and male/female ratio was 40/60%. The prevalence of PCR-confirmed COVID diagnosis was 43.1%. Variplex test was positive in 13.8%. False-negative rate of variplex test compared with PCR was 83.0%. The potential of LAMP technology using isolated RNA has been demonstrated impressively by others, and excellent sensitivity and specificity of detecting SARS-CoV-2 has been reported. However, without RNA extraction, the variplex test system failed to reliably detect SARS-CoV-2 directly in respiratory samples.

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Soroka, Marianna, Barbara Wasowicz, and Anna Rymaszewska. "Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?" Cells 10, no. 8 (July 29, 2021): 1931. http://dx.doi.org/10.3390/cells10081931.

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In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

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Shi, Chao, Fanjin Shang, Meiling Zhou, Pansong Zhang, Yifan Wang, and Cuiping Ma. "Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification." Chemical Communications 52, no. 77 (2016): 11551–54. http://dx.doi.org/10.1039/c6cc05906f.

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Fomicheva, K. A., A. I. Osipyants, M. Y. Shkurnikov, A. A. Pokryschenko, E. A. Tonevitsky, and V. I. Vechorko. "Loop-mediated isothermal amplification holds great potential for massive COVID-19 diagnostics." Biotekhnologiya 36, no. 5 (2020): 6–12. http://dx.doi.org/10.21519/0234-2758-2020-36-5-6-12.

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Real-time RT-PCR currently remains most popular for early COVID-19 diagnostics. However Loop-mediated isothermal amplification (LAMP) method outperform real-time RT-PCR in rapidity and simplicity because it doesn't require expensive laboratory equipment and trained personnel. LAMP-based diagnostic kits for COVID-19 testing are already exist, but LAMP-based tests are not yet widely adopted. The method has great potential for mass application. Here we discuss technical and methodological aspects of its widespread implementation. COVID-19, SARS-CoV-2, LAMP, loop-mediated isothermal amplification, diagnostics, coronavirus. This work was supported by a grant from the President of the Russian Federation for state support of young Russian scientists - candidates of sciences (MK-1906.2019.4).

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Lim, King Ting, Cindy Shuan Ju Teh, and Kwai Lin Thong. "Loop-Mediated Isothermal Amplification Assay for the Rapid Detection ofStaphylococcus aureus." BioMed Research International 2013 (2013): 1–5. http://dx.doi.org/10.1155/2013/895816.

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Staphylococcus aureus, including methicillin-resistantS. aureus(MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting thearcCgene ofS. aureuswas developed and evaluated with 119S. aureusand 25 non-S. aureusstrains. The usefulness of the assay was compared with the PCR method that targetsspaandarcCgenes. The optimal temperature for the LAMP assay was 58.5°C with a detection limit of 2.5 ng/μL and 102 CFU/mL when compared to 12.5 ng/μL and 103 CFU/mL for PCR (spaandarcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureusand one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification ofS. aureus. The LAMP assay is a promising alternative method for the rapid identification ofS. aureusand could be used in resource-limited laboratories and fields.

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Byun, Kye-Hwan, Sang Ha Han, Seungho Choi, Hyeon-Jo Bang, Seong Il Kang, Sookyoung Kim, and Sang-Do Ha. "Comparative efficacy of loop-mediated isothermal amplification, real-time PCR, and selective agar method for detection of Listeria monocytogenes in food." Korean Journal of Food Preservation 29, no. 3 (June 2022): 521–29. http://dx.doi.org/10.11002/kjfp.2022.29.3.521.

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Listeria monocytogenes is a foodborne pathogen causing listeriosis, which can be fatal in specific high-risk groups. The aim of this study was to compare the performance (accuracy, sensitivity, and specificity) of 3M™ Molecular Detection System (3M™ MDS) and Korean Food Codex [real-time PCR (RT-PCR) and selective agar] for the detection of L. monocytogenes in various food matrices. The detection performance of the three methods was determined against 100-103 CFU/mL of L. monocytogenes in vitro and showed high accuracy in the order of RT-PCR, 3M™ MDS, and selective agar. There was no difference in sensitivity and specificity of the three methods. Eleven food matrices, selected from agricultural, livestock, and seafood products, were artificially inoculated with 100-103 CFU/25 g of L. monocytogenes and enriched in 3M™ Demi-Fraser Broth. None of the three methods could completely detect low concentrations of L. monocytogenes in a food matrix. However, 3M™ MDS, which is a loop-mediated isothermal amplification (LAMP)-based technology for rapid detection, showed a higher positive detection rate than RT-PCR did, but lower than that of selective agar. These data indicated that 3M™ MDS was superior for the rapid detection of L. monocytogenes, compared to RT-PCR in food matrices containing various inhibitors. Consequentially, the study findings suggest that the LAMP method is a promising alternative to RT-PCR for the rapid detection of foodborne pathogens.

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Sharafdarkolaee, Somayeh Heidari, Pooria Gill, Majid Motovali-Bashi, and Fatemeh Heidari Sharafdarkolaee. "Isothermal Amplification Methods for the SNP Genotyping." Current Molecular Medicine 19, no. 7 (August 2, 2019): 461–72. http://dx.doi.org/10.2174/1566524019666190527083947.

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The demands for genotyping techniques with acceptable precision, accuracy, cost-effectiveness in high throughput formats made driving forces for continuous development of novel technologies. A wide range of mutation detection techniques based on polymerase chain reaction (PCR) have been introduced. The best alternatives were the isothermal amplification technologies that those did not require a thermal cycler. In this review, we aimed to describe the most known isothermal amplification techniques for SNP genotyping.

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YOKOYAMA, EIJI, MASAKO UCHIMURA, and KENITIRO ITO. "Detection of Enteroaggregative Escherichia coli by Loop-Mediated Isothermal Amplification." Journal of Food Protection 73, no. 6 (June 1, 2010): 1064–72. http://dx.doi.org/10.4315/0362-028x-73.6.1064.

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A novel gene amplification method, loop-mediated isothermal amplification (LAMP), has been recently developed as a rapid, specific diagnostic method for various infectious diseases. We have investigated whether LAMP can be used to detect small numbers of enteroaggregative Escherichia coli (EAEC) cells contaminated in food samples. Primers for LAMP reaction were designed with EAEC aggR gene sequences (available in GenBank). LAMP specificity with these primers was the same as that of PCR in a study of 37 EAEC and 42 non-EAEC bacterial strains. The sensitivity of the LAMP method was better than that of PCR in a study of serially diluted EAEC cells. The LAMP method was significantly more effective than was PCR in detecting EAEC-contaminated food samples (Fisher's exact test, P < 0.05). Therefore, the LAMP method described here should be useful for detecting small numbers of EAEC cells in food samples.

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Buffart, Tineke E., Marianne Tijssen, Thijs Krugers, Beatriz Carvalho, Serge J. Smeets, Ruud H. Brakenhoff, Heike Grabsch, Gerrit A. Meijer, Henry B. Sadowski, and Bauke Ylstra. "DNA Quality Assessment for Array CGH by Isothermal Whole Genome Amplification." Analytical Cellular Pathology 29, no. 4 (January 1, 2007): 351–59. http://dx.doi.org/10.1155/2007/709290.

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Background: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. Material and Methods: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by β-globin PCR. Results: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non-amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. Conclusion: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples.

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Johan, Calvin Wijaya, Sulistyo Emantoko Dwi Putra, and Ernest Suryadjaja. "Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR." KELUWIH: Jurnal Sains dan Teknologi 1, no. 1 (February 28, 2020): 29–37. http://dx.doi.org/10.24123/saintek.v1i1.2775.

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Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi

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Salih, Reem Rabie Mohammed, I. E. T. Aradaib, A. E. Karrar, and H. H. Gibreel. "Loop Mediated Isothermal Amplification Technique (LAMP): A rapid Tool For Detection Of mitochondrial Cytochrome b Gene Of Dorcas gazelle." GABJ 4, no. 1 (December 4, 2021): 22–27. http://dx.doi.org/10.46325/gabj.v4i1.129.

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Loop-mediated isothermal amplification (LAMP) assay was introduced in the year 2000 by Notomi, as a highly sensitive, specific and cost-effective technique for microbial identification. In contrast to the polymerase chain reaction (PCR) technology in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature and does not require a thermal cycler. LAMP, a simple DNA amplification technique, with its field amenable nature has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites and in most of the cases it surpasses polymerase chain reaction. In this study the authors investigated the Loop mediated isothermal amplification technique (LAMP) which is a novel nucleic acid amplification technique. They tried to apply LAMP technique to detection of mitochondrial cytochrome b gene in dorcas gazelles. They designed LAMP specific primers for targeted gene and have verified the LAMP sensitivity up to 4 particles. The authors suggested that LAMP technique could be an appropriate replacement for PCR and may be useful in low resource or field settings where conventional DNA or RNA extraction prior.

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Hussain, Musaddeq. "Isothermal droplet digital PCR method for quantification of CHO residual DNA." Journal of Pharmaceutical and Biomedical Analysis 211 (March 2022): 114564. http://dx.doi.org/10.1016/j.jpba.2021.114564.

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Otgontogtokh, Nyamsuren, Baljidmaa Batmunkh, Bayarmagnai Davganyam, Davaasuren Nergui, Ariunaa Tserendorj, Uyangaa Temuujin, Chimedtseren Bayasgalan, et al. "Preliminary result of insulated isothermal PCR (IIPCR) for avian influenza surveillance." Mongolian Journal of Agricultural Sciences 29, no. 1 (April 30, 2020): 9–14. http://dx.doi.org/10.5564/mjas.v29i1.1364.

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In avian influenza (AI) surveillance, total 686 (fecal n=682, swabs n = 4) samples of migratory birds were collected on August 2018 in Saikhan soum, Bulgan aimag, and these samples were analyzed by portable insulated isothermal (iiPCR) PCR in the field condition. Total of 137 pool of samples (each pool contains 4-6 samples) were analyzed by iiPCR and 3 pools of sample were found positive. Total of 9 pool of samples were positive by virus isolation with embryonated egg inoculation and HA analysis and 4/9 were identified as an avian influenza and 5/9 were identified as a Paramyxovirus by RT-PCR analysis. After sequencing, H3 (n=3) and H2 (n=1) subtypes were determined. iiPCR positive 3 pools were confirmed by egg inoculation and HA analysis and it shows that this newly applied method was able to use to the surveillance of avian influenza due to usefulness and high sensitivity. Шувууны томуугийн тандах судалгаанд зөөврийн полимеразын гинжин урвалыг ашигласан дүнгээс Шувууны томуугийн тандах судалгаанд зөөврийн (insulated isothermal PCR (iiPCR)) ПГУ-ыг хээрийн нөхцөлд хэрэглэх боломжийг турших зориолгоор 2018 оны 8 сард Булган аймгийн Сайхан сум Хунт нуурны орчмоос цуглуулсан нийт 686 (сангас; n=682, арчдас; n=4) усны шувуудын дээжинд хээрийн нөхцөлд шинжилгээ хийж үр дүнг гаргав. Гарсан үр дүнг лабораторид үр хөврөлт өндгөнд өсгөвөрлөх, ЦНУ болон RT-PCR-р баталгаажуулж илэрсэн вирусын НА дэд хэвшлийг нуклеотидын дараалал тогтоох аргаар тодорхойлов. Хээрийн нөхцөлд 137 багц дээжинд (4-6 дээж = 1 багц) iiPCR-р хийсэн илрүүлэх шинжилгээгээр шувууны томуугийн вирусын генийн өвөрмөц хэсэг 3 багц дээжинд илрэв. Лабораторид вирус өсгөвөрлөх болон ЦНУ-р шинжлэхэд 9 багц дээж эерэг гарсанаас шувууны томуугийн вирус (n=4) болон шувууны парамиксовирус (n=5)-ын халдвар байгааг RT-PCR-р баталгаажуулж, гений дараалал тогтоох аргаар шувууны томуугийн H3 (n=3) болон H2 (n=1) дэд хэвшил болохыг тодорхойлов. iiPCR-аар шувууны томуугийн вирусын генийн өвөрмөц хэсэг илэрсэн 3 багц дээжийн үр дүн лабораторийн шинжилгээний дүнгээр баталгаажсан нь энэхүү хэрэглэхэд хялбар, мэдрэг чанар өндөр, түргэвчилсэн аргыг тандах судалгаанд хэрэглэх боломжтойг илтгэж байна. Түлхүүр үг: нүүдлийн шувуу, тандалт, үүсгэгч илрүүлэх, дэд хэвшил

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Paris, Daniel H., Sue J. Lee, Abul M. Faiz, Mahtabuddin Hasan, Mallika Imwong, Nicholas P. J. Day, Arjen M. Dondorp, Emran Bin Yunus, and Kamolrat Silamut. "Loop-Mediated Isothermal PCR (LAMP) for the Diagnosis of Falciparum Malaria." American Journal of Tropical Medicine and Hygiene 77, no. 5 (November 1, 2007): 972–76. http://dx.doi.org/10.4269/ajtmh.2007.77.972.

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Das, Debayan, Manaswini Masetty, and Aashish Priye. "Paper-Based Loop Mediated Isothermal Amplification (LAMP) Platforms: Integrating the Versatility of Paper Microfluidics with Accuracy of Nucleic Acid Amplification Tests." Chemosensors 11, no. 3 (February 28, 2023): 163. http://dx.doi.org/10.3390/chemosensors11030163.

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Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs.

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Zhong, Qing Ping, Li Wang, Bin Wang, and Hong Yuan Chen. "Loop-Mediated Isothermal Amplification Method for Rapid Detection of Shigella dysenteriae." Applied Mechanics and Materials 140 (November 2011): 369–73. http://dx.doi.org/10.4028/www.scientific.net/amm.140.369.

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The study was aimed to develop a loop-mediated isothermal amplification (LAMP) method which amplifies DNA with high specificity and rapidity for the detection of Shigella dysenteriae. A set of four primers was designed for recognizing six distinct sequences on the target ipaH of S. dysenteriae. By the method, the target DNA was amplified within 1h under isothermal condition at 65 °C. The sensitivities of the LAMP for detecting pure culture and genomic DNA were 1.04 CFU/ml and 1.06 fg/μl, while the sensitivities of PCR method were 1.04×102 CFU/ml and 1.06 pg/μl. Furthermore, the LAMP assay was examined for its ability to detect S. dysenteriae in artificially contaminated lettuce sample, the detection limits of this LAMP assay and the PCR method were 4.60 CFU/g and 4.60×102 CFU/g, respectively.

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Murwantoko, Murwantoko. "Metode Loop-Mediated Isothermal Amplification (LAMP) dan Aplikasinya untuk Deteksi Penyakit Ikan." Jurnal Perikanan Universitas Gadjah Mada 8, no. 1 (February 15, 2006): 1. http://dx.doi.org/10.22146/jfs.156.

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Polymerase chain reaction (PCR) is a method that amplifies DNA which have been widely used in molecular biology technique. Based on the PCR, many methods have been developed on isothermal condition and the useful one is loop-mediated isothermal amplification of DNA (LAMP). LAMP reaction employs a Bst DNA polymerase and a set of four specific primers that recognizes a total of six distinct sequences of the target DNA and produces amount of different size of DNA. Many advantages have been achieved in LAMP such as the simple equipment for reaction and observation, short time, highly specific and sensitive procedure. LAMP has been used as a tools for detection many pathogens for human, animals and plants. Some fish pathogens as parasites, bacteria and viruses have been detected by LAMP. The principles and application of LAMP method are discussed in this paper.

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PARTHIBAN, M., K. S. SHALINI, KURUNCHI C. DIVYA, K. KUMANAN, and K. S. AARTHI. "Rapid detection of fowl adenovirus field samples using loop-mediated isothermal amplification assay." Indian Journal of Animal Sciences 84, no. 1 (January 30, 2014): 22–25. http://dx.doi.org/10.56093/ijans.v84i1.37295.

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Loop-mediated isothermal amplification was employed for the detection of fowl adenoviruses as a simple, rapid and field based diagnostic assay. Out of 134 field samples screened, 28 samples were positive by both PCR and LAMP assay. The LAMP assay primers were found to be highly specific and it detected only fowl adenovirus samples and it did not react with other avian viruses like Marek’s disease virus and avian leucosis virus. Conventional PCR detected 100ug level of DNA whereas LAMP assay detected up to 10ng level of DNA. It clearly indicated that LAMP assay was 100-times more sensitive than conventional PCR. The specificity of LAMP assay was confirmed by sequencing of the LAMP amplified products and real time PCR. The LAMP positive reaction can be easily visualized under UV trans- illuminator by the addition of SYBR green dye. Thus, LAM Passay proves to be a sensitive, rapid, less-laborious and cost- effective technique for the diagnosis of fowl adenoviruses in field conditions.

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Boonbanjong, Poramin, Kiatnida Treerattrakoon, Wassa Waiwinya, Piyawat Pitikultham, and Deanpen Japrung. "Isothermal Amplification Technology for Disease Diagnosis." Biosensors 12, no. 9 (August 24, 2022): 677. http://dx.doi.org/10.3390/bios12090677.

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Isothermal amplification (IA) is a nucleic acid amplification technology (NAAT) that has contributed significantly to the healthcare system. The combination of NAAT with a suitable detection platform resulted in higher sensitivity, specificity, and rapid disease diagnosis. Traditional NAAT, such as polymerase chain reaction (PCR), is widely applied in the general healthcare system but is rarely accessed in resource-limited hospitals. Some IA methods provide a rapid, sensitive, specific, and simple method for disease diagnosis. However, not all IA techniques have been regularly used in clinical applications because different biomarkers and sample types affect either the enzyme in the IA system or sample preparation. This review focuses on the application of some IA techniques that have been applied in the medical field and have the potential for use at points of care.

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Yusop, M. H. M., and M. F. A. Bakar. "Review on halal forensic: a focus on DNA-based methods for pork authentication." Food Research 4, no. 6 (August 20, 2020): 2347–54. http://dx.doi.org/10.26656/fr.2017.4(6).180.

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Food product authentication is important at every level of the food manufacturing process, starting from raw materials until finished products. Authentication also plays an important role in assuring accurate food labelling, which is required to help consumers select suitable types of food products. Food adulteration is one of the vital issues addressed by halal authentication, especially for food products that contain pig traces or porcine ingredients. Various methods that aim to guarantee the authenticity of foods have been developed over the past years. In this article, a short review of recent food analytical methods related to authenticity studies, with special regard to pork identification, is provided. The focus of this review is DNA-based methods, which have gained the interest of the scientific community. The specificity, sensitivity and fast and high throughput of the methods are highlighted. In the present case, methods that are capable of detecting pork by using DNA barcode, polymerase chain reaction (PCR)-restriction fragment length polymorphism, conventional PCR, real-time PCR and isothermal amplification are discussed. Although PCR is the most popular method, recent studies have shown that isothermal amplification is a potential alternative because it is rapid, simple and does not require the use of any complicated instruments, such as a thermal cycler and sequencer.

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Shiluli, Clement, Shwetha Kamath, Bernard N. Kanoi, Racheal Kimani, Michael Maina, Harrison Waweru, Moses Kamita, et al. "Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis." Open Research Africa 7 (May 9, 2024): 2. http://dx.doi.org/10.12688/openresafrica.14316.2.

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Chlamydia trachomatis (C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/μL to 1×10-3pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

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Shiluli, Clement, Shwetha Kamath, Bernard N. Kanoi, Racheal Kimani, Michael Maina, Harrison Waweru, Moses Kamita, et al. "Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis." Open Research Africa 7 (January 8, 2024): 2. http://dx.doi.org/10.12688/openresafrica.14316.1.

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Chlamydia trachomatis (C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/mL to 1×10-3pg/mL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

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Garneret, Pierre, Etienne Coz, Elian Martin, Jean-Claude Manuguerra, Elodie Brient-Litzler, Vincent Enouf, Daniel Felipe González Obando, et al. "Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification." PLOS ONE 16, no. 1 (January 11, 2021): e0243712. http://dx.doi.org/10.1371/journal.pone.0243712.

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To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT—PCR (Reverse transcription—Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT—LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross‐reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called “COVIDISC” to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

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Moon, Ye-Ji, So-Young Lee, and Se-Wook Oh. "A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens." Foods 11, no. 3 (January 24, 2022): 322. http://dx.doi.org/10.3390/foods11030322.

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The isothermal amplification method, a molecular-based diagnostic technology, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), is widely used as an alternative to the time-consuming and labor-intensive culture-based detection method. However, food matrices or other compounds can inhibit molecular-based diagnostic technologies, causing reduced detection efficiencies, and false-negative results. These inhibitors originating from food are polysaccharides and polyphenolic compounds in berries, seafood, and vegetables. Additionally, magnesium ions needed for amplification reactions can also inhibit molecular-based diagnostics. The successful removal of inhibitors originating from food and molecular amplification reaction is therefore proposed to enhance the efficiency of molecular-based diagnostics and allow accurate detection of food-borne pathogens. Among molecular-based diagnostics, PCR inhibitors have been reported. Nevertheless, reports on the mechanism and removal of isothermal amplification method inhibitors are insufficient. Therefore, this review describes inhibitors originating from food and some compounds inhibiting the detection of food-borne pathogens during isothermal amplification.

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Lee, David, Manuela Ialicicco, Harika Akkinepalli, Giacomo Morreale, Yan Liu, Hei Leung, Gabriella S. Scippa, Andy Greenland, and Ian Mackay. "Genotyping SSR length variants by isothermal DNA amplification." Genome 55, no. 09 (September 2012): 691–95. http://dx.doi.org/10.1139/g2012-058.

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Loop-mediated isothermal DNA amplification (LAMP) is an alternative method for the amplification of DNA sequences. It has been applied primarily for the detection of specific targets. We demonstrate the novel use of LAMP to amplify SSR alleles in a set of rice varieties and show the results to be consistent with analysis performed by PCR. Furthermore, we test the sensitivity of the assay and show it to amplify from near single copy target.

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Lung, O., J. Pasick, M. Fisher, C. Buchanan, A. Erickson, and A. Ambagala. "Insulated Isothermal Reverse Transcriptase PCR (iiRT-PCR) for Rapid and Sensitive Detection of Classical Swine Fever Virus." Transboundary and Emerging Diseases 63, no. 5 (January 27, 2015): e395-e402. http://dx.doi.org/10.1111/tbed.12318.

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Jones, Laura M., Anthony J. Flemming, and Peter E. Urwin. "NHR-176 regulates cyp-35d1 to control hydroxylation-dependent metabolism of thiabendazole in Caenorhabditis elegans." Biochemical Journal 466, no. 1 (February 6, 2015): 37–44. http://dx.doi.org/10.1042/bj20141296.

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This study combines metabolite and phenotype analyses in conjunction with RNAi, quantitative PCR and isothermal titration calorimetry to determine the precise metabolic pathway of a widely used anthelmintic in Caenorhabditis elegans. We demonstrate the presence of a metabolite unique to nematodes.

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Byung-Yong, PARK, SHIM Kwan-Seob, KIM Won-Il, HOSSAIN Md Mukter, KIM Bumseok, KWON Jungkee, PARK Choi-Kyu, CHO Sung-Jin, JO Inho, and CHO Ho-Seong. "Rapid And Sensitive Detection Of Lawsonia Intracellularis In Pigs By Real-Time Loop-Mediated Isothermal Amplification." Acta Veterinaria 65, no. 1 (March 1, 2015): 20–29. http://dx.doi.org/10.1515/acve-2015-0002.

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Abstract A simple and rapid real-time loop-mediated isothermal amplification (LAMP) assay designed to detect Lawsonia (L.) intracellularis, an important bacteria causing proliferative enteropathy in pigs. A set of four primers targeting the ubiquinone/menaquinone biosynthesis methylase (ubiE) gene was designed for the LAMP reaction. Additionally, serial 10-fold dilutions of cultured L. intracellularis and spiked feces were also used for the optimization of real-time LAMP. The lower limit of the linear range of the assay in L. intracellularis was 1.0 × 100 L. intracellularis. Real-time LAMP was 10 and 100 times more sensitive than real-time PCR and conventional PCR detection methods, respectively. Based on testing of 213 porcine fecal samples using real-time LAMP, realtime PCR and PCR, the agreement quotients of real-time LAMP with conventional PCR and with real-time PCR were 0.77 and 0.95, respectively. This study demonstrated that real-time LAMP was a powerful tool for the rapid and sensitive detection of L. intracellularis in porcine fecal samples.

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Feranisa, Anggun. "KOMPARASI ANTARA POLYMERASE CHAIN REACTION (PCR) DAN LOOPMEDIATED ISOTHERMAL AMPLIFICATION (LAMP) DALAM DIAGNOSIS MOLEKULER." ODONTO : Dental Journal 3, no. 2 (December 1, 2016): 145. http://dx.doi.org/10.30659/odj.3.2.145-151.

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Background: Molecular diagnostic is an emerging diagnostic method inpersonalized medicine/dentistry era. Usually, it uses nucleic acid amplificationmethod to detect various diseases. PCR is conventional nucleic acid amplification method. However, due to an urgency in infectious diseases’ diagnotic method, scientists developed LAMP as new nucleic acid amplification method.Discussion: There are various experiments used to develop LAMP as infectious diseases diagnostic method compared to PCR. The results are LAMP more sensitive, specific, rapid, and inexpensive than PCR.Conclusion: Both PCR and LAMP can be used as molecular diagnostic tools.LAMP prefer to used as infectious disease diagnostic method in poor anddeveloping countries.

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Areekit, Supatra, Pongbun Tangjitrungrot, Sintawee Khuchareontaworn, Kankanit Rattanathanawan, Pornpun Jaratsing, Montri Yasawong, Gaysorn Chansiri, Nareerat Viseshakul, and Kosum Chansiri. "Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2)." Current Issues in Molecular Biology 44, no. 11 (November 3, 2022): 5427–39. http://dx.doi.org/10.3390/cimb44110368.

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Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

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Antropov, Denis N., and Grigory A. Stepanov. "Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection." Current Issues in Molecular Biology 45, no. 1 (January 10, 2023): 649–62. http://dx.doi.org/10.3390/cimb45010043.

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Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.

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Ngoc, Le Thi Nhu, and Young-Chul Lee. "Current Trends in RNA Virus Detection via Nucleic Acid Isothermal Amplification-Based Platforms." Biosensors 14, no. 2 (February 11, 2024): 97. http://dx.doi.org/10.3390/bios14020097.

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Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

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García-Bernalt Diego, Juan, Pedro Fernández-Soto, and Antonio Muro. "LAMP in Neglected Tropical Diseases: A Focus on Parasites." Diagnostics 11, no. 3 (March 15, 2021): 521. http://dx.doi.org/10.3390/diagnostics11030521.

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Neglected Tropical Diseases (NTDs), particularly those caused by parasites, remain a major Public Health problem in tropical and subtropical regions, with 10% of the world population being infected. Their management and control have been traditionally hampered, among other factors, by the difficulty to deploy rapid, specific, and affordable diagnostic tools in low resource settings. This is especially true for complex PCR-based methods. Isothermal nucleic acid amplification techniques, particularly loop-mediated isothermal amplification (LAMP), appeared in the early 21st century as an alternative to PCR, allowing for a much more affordable molecular diagnostic. Here, we present the status of LAMP assays development in parasite-caused NTDs. We address the progress made in different research applications of the technique: xenomonitoring, epidemiological studies, work in animal models and clinical application both for diagnosis and evaluation of treatment success. Finally, we try to shed a light on the improvements needed to achieve a true point-of-care test and the future perspectives in this field.

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Wibowo, Muhammad Rizky, Erna Harfiani, Sarmoko Sarmoko, and Yudhi Nugraha. "The detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP) in developing country." Pharmacy Reports 1, no. 1 (August 31, 2021): 9. http://dx.doi.org/10.51511/pr.9.

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The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected the human system resulting in Covid-19, and has spread rapidly worldwide. Therefore, a fast, simple, cost-effective, and accurate detecting tool is required. The standard diagnostic tool of the World Health Organization is the reverse transcription-polymerase chain reaction (RT-PCR). This method detects the presence of viral genetic material in the human body with accurate results. However, it has several limitations in terms of equipment, personnel, duration, and cost. Therefore, a fast, simple, and sensitive alternative detection, is required, one of which is the reverse transcription loop-mediated isothermal amplification (RT-LAMP) that functions under isothermal conditions. This method is battery-driven, hence, easy to move closer to the patient. Conclusively, the RT-LAMP test for SARS CoV-2 diagnosis produces comparable sensitivity to a standard RT-PCR and is more suitable for resource-poor settings, such as rural areas of developing countries.

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Wang, De Guo. "Rapid Detection of Mycobacterium tuberculosis Complex by Loop-Mediated Isothermal Amplification Combined with Chemosensor." Advanced Materials Research 749 (August 2013): 449–52. http://dx.doi.org/10.4028/www.scientific.net/amr.749.449.

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Loop-mediated isothermal amplification (LAMP) allowed rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chemosensor for much more efficient, field-friendly detection of Mycobacterium tuberculosis complex. In this report, LAMP was performed at 65 °C for 10 min, followed by a rapid reaction of DNA amplification by-product, pyrophosphate ion, with chemosensor resulted in red disappearance. The detection limit of Mycobacterium tuberculosis complex by LAMP-Chemosensor was 3-5 copies, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with Real-time PCR. The results showed that the LAMP-Chemosensor method had the advantages of better sensitivity and speed and less dependence on equipment than the standard (PCR) for specifically detecting low levels of Mycobacterium tuberculosis complex DNA, and this can be useful in the field as a routine diagnostic tool.

Journal articles on the topic 'Isothermal pcr'

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