Статті в журналах: "Nucleic-acid Amplification and Quantification"

1

Huggett, Jim, Clare Green, and Alimuddin Zumla. "Nucleic acid detection and quantification in the developing world." Biochemical Society Transactions 37, no. 2 (March 20, 2009): 419–23. http://dx.doi.org/10.1042/bst0370419.

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Techniques using nucleic acid amplification have not had the same amount of impact on research and clinical diagnosis in the developing world as that observed in the West. This is unsurprising when the costs and infrastructure required to perform nucleic acid amplification are considered. Despite this, nucleic acid amplification is being increasingly used in both research and diagnosis in countries such as Zambia and Tanzania. Scientific research in the developing world is made possible through the support and development of the necessary laboratory infrastructure and the establishment of special transport for the reagents and samples. This has enabled world-leading country-relevant research to be performed by local scientists on subjects ranging from rapid diagnosis of infectious diseases to measuring the RNA gene expression in an immune response. Concomitantly, the challenge presented by the need for tests that are more appropriate for a resource-poor setting has led to a number of newer methodologies for nucleic acid detection, which can be tailored to be performed in the field without the need for training in molecular biology. As nucleic acid amplification techniques become both simpler and cheaper, their impact is likely to play an increasingly crucial role in research and diagnosis in the developing world.

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Kreutz, Jason E., Jiasi Wang, Allison M. Sheen, Alison M. Thompson, Jeannette P. Staheli, Michael R. Dyen, Qinghua Feng, and Daniel T. Chiu. "Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP." Lab on a Chip 19, no. 6 (2019): 1035–40. http://dx.doi.org/10.1039/c8lc01223g.

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3

Becherer, Lisa, Jacob Friedrich Hess, Sieghard Frischmann, Mohammed Bakheit, Hans Nitschko, Silvina Stinco, Friedrich Zitz, et al. "Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP." Micromachines 12, no. 2 (February 5, 2021): 159. http://dx.doi.org/10.3390/mi12020159.

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This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

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4

Mauk, Michael, Changchun Liu, Jinzhao Song, and Haim Bau. "Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification." Microarrays 4, no. 4 (October 20, 2015): 474–89. http://dx.doi.org/10.3390/microarrays4040474.

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5

Gullett, Jonathan C., and Frederick S. Nolte. "Quantitative Nucleic Acid Amplification Methods for Viral Infections." Clinical Chemistry 61, no. 1 (January 1, 2015): 72–78. http://dx.doi.org/10.1373/clinchem.2014.223289.

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AbstractBACKGROUNDOver the past 2 decades there have been substantial improvements in the methods used to quantify viral nucleic acid in body fluids and in our understanding of how to use viral load measurements in the diagnosis and management of patients with a number of viral infections. These methods are now integrated into a wide range of diagnostic and treatment guidelines and commonly deployed in a variety of clinical settings.CONTENTQuantitative nucleic acid amplification methods that are used to measure viral load are described along with key issues and important variables that affect their performance. Particular emphasis is placed on those methods used in clinical laboratories as US Food and Drug Administration–cleared or laboratory-developed tests. We discuss the clinical applications of these methods in patients with HIV-1, hepatitis C virus, hepatitis B virus, cytomegalovirus, Epstein-Barr virus, and BK polyomavirus infections. Finally, the current challenges and future directions of viral load testing are examined.SUMMARYQuantitative nucleic acid amplification tests provide important information that can be used to predict disease progression, distinguish symptomatic from asymptomatic infection, and assess the efficacy of antiviral therapy. Despite the advances in technology, large challenges remain for viral testing related to accuracy, precision, and standardization. Digital PCR, a direct method of quantification of nucleic acids that does not rely on rate-based measurements or calibration curves, may address many of the current challenges.

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6

Zhang, Min, Jiajia Wu, Zhaoai Shi, Aocheng Cao, Wensheng Fang, Dongdong Yan, Qiuxia Wang, and Yuan Li. "Molecular Methods for Identification and Quantification of Foodborne Pathogens." Molecules 27, no. 23 (November 26, 2022): 8262. http://dx.doi.org/10.3390/molecules27238262.

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Foodborne pathogens that enter the human food chain are a significant threat worldwide to human health. Timely and cost-effective detection of them became challenging for many countries that want to improve their detection and control of foodborne illness. We summarize simple, rapid, specific, and highly effective molecular technology that is used to detect and identify foodborne pathogens, including polymerase chain reaction, isothermal amplification, loop-mediated isothermal amplification, nucleic acid sequence-based amplification, as well as gene chip and gene probe technology. The principles of their operation, the research supporting their application, and the advantages and disadvantages of each technology are summarized.

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Kurn, Nurith, Pengchin Chen, Joe Don Heath, Anne Kopf-Sill, Kathryn M. Stephens, and Shenglong Wang. "Novel Isothermal, Linear Nucleic Acid Amplification Systems for Highly Multiplexed Applications." Clinical Chemistry 51, no. 10 (October 1, 2005): 1973–81. http://dx.doi.org/10.1373/clinchem.2005.053694.

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Abstract Background: Global analysis of the genome, transcriptome, and proteome is facilitated by the recent development of tools for large-scale, highly parallel analysis. We describe a novel nucleic acid amplification system that generates products by several methods. 3′-Ribo-SPIA™ primes cDNA synthesis at the 3′ polyA tail, and whole transcript (WT)-Ribo-SPIA primes cDNA synthesis across the full length of the transcripts and thus provides whole-transcriptome amplification, independent of the 3′ polyA tail. Methods: We developed isothermal linear nucleic acid amplification systems, which use a single chimeric primer, for amplification of DNA (SPIA) and RNA (Ribo-SPIA). The latter allows mRNA amplification from as little as 1 ng of total RNA. Amplification efficiency was calculated based on the delta threshold cycle between nonamplified cDNA targets and amplified cDNA. The amounts and quality of total RNA and amplification products were determined after purification of the amplification products. GeneChip® array gene expression profiling and real-time PCR were used to test the accuracy and reproducibility of the method. Quantification of cDNA products (before and after amplification) at the 2 loci along the transcripts was used to assess product length (for evaluation of the 3′-initiated Ribo-SPIA) and equal representation throughout the length of the transcript (for evaluation of the whole transcript amplification system, WT-Ribo-SPIA™). Results: Ribo-SPIA–based global RNA amplification exhibited linearity over 6 orders of magnitude of transcript abundance and generated microgram amounts of amplified cDNA from as little as 1 ng of total RNA. Conclusions: The described methods enable comprehensive gene expression profiling and analysis from limiting biological samples. The WT-Ribo-SPIA procedure, which enables amplification of non–polyA-tailed RNA, is suitable for amplification and gene expression analysis of both eukaryotic and prokaryotic biological samples.

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Morcia, Caterina, Roberta Ghizzoni, Chiara Delogu, Lorella Andreani, Paola Carnevali, and Valeria Terzi. "Digital PCR: What Relevance to Plant Studies?" Biology 9, no. 12 (November 30, 2020): 433. http://dx.doi.org/10.3390/biology9120433.

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Digital PCR (dPCR) is a breakthrough technology that able to provide sensitive and absolute nucleic acid quantification. It is a third-generation technology in the field of nucleic acid amplification. A unique feature of the technique is that of dividing the sample into numerous separate compartments, in each of which an independent amplification reaction takes place. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for reading the digital output data. The dPCR assays developed so far in the plant science sector were identified in the literature, and the major applications, advantages, disadvantages, and applicative perspectives of the technique are presented and discussed in this review.

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9

Warrior, Usha, Yihong Fan, Caroline A. David, Julie A. Wilkins, Evelyn M. McKeegan, James L. Kofron, and David J. Burns. "Application of QuantiGene™ Nucleic Acid Quantification Technology for High Throughput Screening." Journal of Biomolecular Screening 5, no. 5 (October 2000): 343–51. http://dx.doi.org/10.1177/108705710000500506.

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To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the Quanti-Gene™ nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-la (IL-la) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.

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10

Li, Xue-Mei, Jie Luo, Ning-Bo Zhang, and Qing-Li Wei. "Nucleic acid quantification using nicking–displacement, rolling circle amplification and bio-bar-code mediated triple-amplification." Analytica Chimica Acta 881 (June 2015): 117–23. http://dx.doi.org/10.1016/j.aca.2015.05.011.

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11

Patterson, Adriana S., Kuangwen Hsieh, H. Tom Soh, and Kevin W. Plaxco. "Electrochemical real-time nucleic acid amplification: towards point-of-care quantification of pathogens." Trends in Biotechnology 31, no. 12 (December 2013): 704–12. http://dx.doi.org/10.1016/j.tibtech.2013.09.005.

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12

Schouten, J. P. "Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification." Nucleic Acids Research 30, no. 12 (June 15, 2002): 57e—57. http://dx.doi.org/10.1093/nar/gnf056.

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13

Chiorcea-Paquim, Ana-Maria. "Advances in Electrochemical Biosensor Technologies for the Detection of Nucleic Acid Breast Cancer Biomarkers." Sensors 23, no. 8 (April 20, 2023): 4128. http://dx.doi.org/10.3390/s23084128.

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Breast cancer is the second leading cause of cancer deaths in women worldwide; therefore, there is an increased need for the discovery, development, optimization, and quantification of diagnostic biomarkers that can improve the disease diagnosis, prognosis, and therapeutic outcome. Circulating cell-free nucleic acids biomarkers such as microRNAs (miRNAs) and breast cancer susceptibility gene 1 (BRCA1) allow the characterization of the genetic features and screening breast cancer patients. Electrochemical biosensors offer excellent platforms for the detection of breast cancer biomarkers due to their high sensitivity and selectivity, low cost, use of small analyte volumes, and easy miniaturization. In this context, this article provides an exhaustive review concerning the electrochemical methods of characterization and quantification of different miRNAs and BRCA1 breast cancer biomarkers using electrochemical DNA biosensors based on the detection of hybridization events between a DNA or peptide nucleic acid probe and the target nucleic acid sequence. The fabrication approaches, the biosensors architectures, the signal amplification strategies, the detection techniques, and the key performance parameters, such as the linearity range and the limit of detection, were discussed.

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Todd, Alison V., Caroline J. Fuery, Helen L. Impey, Tanya L. Applegate, and Margaret A. Haughton. "DzyNA-PCR: Use of DNAzymes to Detect and Quantify Nucleic Acid Sequences in a Real-Time Fluorescent Format." Clinical Chemistry 46, no. 5 (May 1, 2000): 625–30. http://dx.doi.org/10.1093/clinchem/46.5.625.

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Abstract Background: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum. Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.

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Wang, Jiasi, Jason E. Kreutz, Alison M. Thompson, Yuling Qin, Allison M. Sheen, Jingang Wang, Li Wu, et al. "SD-chip enabled quantitative detection of HIV RNA using digital nucleic acid sequence-based amplification (dNASBA)." Lab on a Chip 18, no. 22 (2018): 3501–6. http://dx.doi.org/10.1039/c8lc00956b.

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Iwanaga, Masanobu. "Rapid Detection of Attomolar SARS-CoV-2 Nucleic Acids in All-Dielectric Metasurface Biosensors." Biosensors 12, no. 11 (November 8, 2022): 987. http://dx.doi.org/10.3390/bios12110987.

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Worldwide infection due to SARS-CoV-2 revealed that short-time and extremely high-sensitivity detection of nucleic acids is a crucial technique for human beings. Polymerase chain reactions have been mainly used for the SARS-CoV-2 detection over the years. However, an advancement in quantification of the detection and shortening runtime is important for present and future use. Here, we report a rapid detection scheme that is a combination of nucleic acid amplification and a highly efficient fluorescence biosensor, that is, a metasurface biosensor composed of a pair of an all-dielectric metasurface and a microfluidic transparent chip. In the present scheme, we show a series of proof-of-concept experimental results that the metasurface biosensors detected amplicons originating from attomolar SARS-CoV-2 nucleic acids and that the amplification was implemented within 1 h. Furthermore, this detection capability substantially satisfies an official requirement of 100 RNA copies/140 μL, which is a criterion for the reliable infection tests.

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Cairns, Murray J., Rachel Turner, and Lun-Quan Sun. "Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion." Biochemical and Biophysical Research Communications 318, no. 3 (June 2004): 684–90. http://dx.doi.org/10.1016/j.bbrc.2004.04.077.

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Oliveira, Beatriz, Bruno Veigas, Alexandra R. Fernandes, Hugo Águas, Rodrigo Martins, Elvira Fortunato, and Pedro Viana Baptista. "Fast Prototyping Microfluidics: Integrating Droplet Digital Lamp for Absolute Quantification of Cancer Biomarkers." Sensors 20, no. 6 (March 14, 2020): 1624. http://dx.doi.org/10.3390/s20061624.

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Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in “Shrinky-dinks” polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

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Zhao, Yue, Huaqing Liu, Feng Chen, Min Bai, and Yongxi Zhao. "Fidelity quantification of mercury(ii) ion via circumventing biothiols-induced sequestration in enzymatic amplification system." RSC Advances 6, no. 83 (2016): 80296–301. http://dx.doi.org/10.1039/c6ra16960k.

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Zhao, Yue, Feng Chen, Jing Qin, Jing Wei, Wenhua Wu, and Yongxi Zhao. "Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples." Chemical Science 9, no. 2 (2018): 392–97. http://dx.doi.org/10.1039/c7sc03994h.

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Schoone, Gerard J., Linda Oskam, Nel C. M. Kroon, Henk D. F. H. Schallig, and Sabah A. Omar. "Detection and Quantification of Plasmodium falciparumin Blood Samples Using Quantitative Nucleic Acid Sequence-Based Amplification." Journal of Clinical Microbiology 38, no. 11 (2000): 4072–75. http://dx.doi.org/10.1128/jcm.38.11.4072-4075.2000.

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A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 108 Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.

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Nicolini, Ariana M., Tyler D. Toth, Samuel Y. Kim, M. Alejandra Mandel, David W. Galbraith, and Jeong-Yeol Yoon. "Mie Scatter and Interfacial Tension Based Real-Time Quantification of Colloidal Emulsion Nucleic Acid Amplification." Advanced Biosystems 1, no. 10 (September 7, 2017): 1700098. http://dx.doi.org/10.1002/adbi.201700098.

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23

Abardía-Serrano, Clara, Rebeca Miranda-Castro, Noemí de-los-Santos-Álvarez, and María Jesús Lobo-Castañón. "New Uses for the Personal Glucose Meter: Detection of Nucleic Acid Biomarkers for Prostate Cancer Screening." Sensors 20, no. 19 (September 26, 2020): 5514. http://dx.doi.org/10.3390/s20195514.

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A personal glucose meter (PGM)-based method for quantitative detection of a urinary nucleic acid biomarker in prostate cancer screening, the so-called PCA3, is reported herein. A sandwich-type genoassay is conducted on magnetic beads to collect the target from the sample by specific hybridization, making the assay appropriate for PCA3 detection in biological fluids. The success of the method hinges on the use of alkaline phosphatase (ALP) to link the amount of nucleic acid biomarker to the generation of glucose. In particular, specifically attached ALP molecules hydrolyze D-glucose-1-phosphate into D-glucose, thus enabling the amplification of the recorded signal on the personal glucose meter. The developed genoassay exhibits good sensitivity (3.3 ± 0.2 mg glucose dL−1 pM−1) for PCA3, with a dynamic range of 5 to 100 pM and a quantification limit of 5 pM. Likewise, it facilitates point-of-care testing of nucleic acid biomarkers by using off-the-shelf PGM instead of complex instrumentation involved in traditional laboratory-based tests.

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Abd El Galil, Khaled H., M. A. El Sokkary, S. M. Kheira, Andre M. Salazar, Marylynn V. Yates, Wilfred Chen, and Ashok Mulchandani. "Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus." Applied and Environmental Microbiology 71, no. 11 (November 2005): 7113–16. http://dx.doi.org/10.1128/aem.71.11.7113-7116.2005.

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ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

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Chen, Jiyun, Xiaomin Xu, Zhimei Huang, Yuan Luo, Lijuan Tang, and Jian-Hui Jiang. "BEAMing LAMP: single-molecule capture and on-bead isothermal amplification for digital detection of hepatitis C virus in plasma." Chemical Communications 54, no. 3 (2018): 291–94. http://dx.doi.org/10.1039/c7cc08403j.

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A novel dNAD platform BEAMing LAMP is developed by combining emulsion micro-reactors, single-molecule magnetic capture and on-bead amplification for absolute and high-precision quantification of nucleic acids.

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Uhd, Jesper, Laura Miotke, Hanlee P. Ji, Marina Dunaeva, Ger J. M. Pruijn, Christian Damsgaard Jørgensen, Emil Laust Kristoffersen, et al. "Ultra-fast detection and quantification of nucleic acids by amplification-free fluorescence assay." Analyst 145, no. 17 (2020): 5836–44. http://dx.doi.org/10.1039/d0an00676a.

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Collins, M. "A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml." Nucleic Acids Research 25, no. 15 (August 1, 1997): 2979–84. http://dx.doi.org/10.1093/nar/25.15.2979.

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Casper, Erica T., John H. Paul, Matthew C. Smith, and Michael Gray. "Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification." Applied and Environmental Microbiology 70, no. 8 (August 2004): 4727–32. http://dx.doi.org/10.1128/aem.70.8.4727-4732.2004.

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ABSTRACT Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

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Sunshine, Andrew Michael, Sarah Ake, and Gangli Wang. "One-Step Attomolar Quantification of Sars-CoV2 RNAs with Aptamer-Based Electrochemical Sensors." ECS Meeting Abstracts MA2022-02, no. 63 (October 9, 2022): 2627. http://dx.doi.org/10.1149/ma2022-02632627mtgabs.

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Reliable quantification of SARS-CoV2 RNA in clinical samples presents an urgent challenge for biosensor technologies. Current benchmark methods are based on rt-qPCR technique which involve isolating RNA from samples, amplifying target RNA, then signal readout. Viral loads are reported in terms of Cq-- the number of PCR cycles before the target concentration is detectable. However, variability in instruments and methodology and a lack of standardization complicates the translation of Cq values into meaningful quantitation such as target concentration in the sample. A viral quantification has the potential to better inform prognoses, enhance treatment protocols, and improve population monitoring via waste water sampling. Herein, an aptamer-based electrochemical sensor is developed. In stark contrast to the almost exclusively adopted sample amplifications in PCR and other fast testing tools, in-situ signal amplification based on redox cycling enables one-step quantification of SARS-CoV2 RNA concentration. Signal-on mechanism and sensor specificity are based on the re-folding of nucleic acid probes in complementary to the target RNA sequence/s which alter the proximity of a redox probe to the gold microelectrodes. Initial results suggest the sensors detects viral RNA concentrations as low as 10 aM within minutes. Ongoing work is focused on procedural optimizations and reproducibility.

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Schneider, Petra, Gerard Schoone, Henk Schallig, Danielle Verhage, Denise Telgt, Wijnand Eling, and Robert Sauerwein. "Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification." Molecular and Biochemical Parasitology 137, no. 1 (September 2004): 35–41. http://dx.doi.org/10.1016/j.molbiopara.2004.03.018.

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Rudi, Knut, Olav M. Skulberg, Frank Larsen, and Kjetill S. Jakobsen. "Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes." Applied and Environmental Microbiology 64, no. 7 (July 1, 1998): 2639–43. http://dx.doi.org/10.1128/aem.64.7.2639-2643.1998.

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ABSTRACT A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genusMicrocystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

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Zhang, Zengming, Shuhao Zhao, Fei Hu, Guangpu Yang, Juan Li, Hui Tian, and Niancai Peng. "An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification." Micromachines 11, no. 2 (February 8, 2020): 177. http://dx.doi.org/10.3390/mi11020177.

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The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/μL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.

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33

Kankia, Besik. "Isothermal Amplification and Quantification of Nucleic Acids Using Intrinsic Fluorescence of Primers." Biophysical Journal 98, no. 3 (January 2010): 63a. http://dx.doi.org/10.1016/j.bpj.2009.12.358.

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34

Naka, Norifumi, Susumu Joyama, Yoshitane Tsukamoto, Kiyoko Yoshioka, Nobuyuki Hashimoto, Takeshi Ujiiye, Tsukasa Hayashi, et al. "Quantification of SSX mRNA Expression in Human Bone and Soft Tissue Tumors Using Nucleic Acid Sequence-Based Amplification." Journal of Molecular Diagnostics 7, no. 2 (May 2005): 187–97. http://dx.doi.org/10.1016/s1525-1578(10)60545-4.

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35

Jespers, Vicky, Joris Menten, Hilde Smet, Sabrina Poradosú, Saïd Abdellati, Rita Verhelst, Liselotte Hardy, Anne Buvé, and Tania Crucitti. "Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests." BMC Microbiology 12, no. 1 (2012): 83. http://dx.doi.org/10.1186/1471-2180-12-83.

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Hayashi, Tsukasa, Hiroyuki Kobayashi, Hayato Miyachi, Toshio Ohshima, Takeshi Ujiiye, Masako Kawase, Tomomitsu Hotta, and Yuzuru Takemura. "A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells." Clinica Chimica Acta 342, no. 1-2 (April 2004): 115–26. http://dx.doi.org/10.1016/j.cccn.2003.12.013.

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37

Schuler, Friedrich, Frank Schwemmer, Martin Trotter, Simon Wadle, Roland Zengerle, Felix von Stetten, and Nils Paust. "Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA." Lab on a Chip 15, no. 13 (2015): 2759–66. http://dx.doi.org/10.1039/c5lc00291e.

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38

Schneider, P., L. Wolters, G. Schoone, H. Schallig, P. Sillekens, R. Hermsen, and R. Sauerwein. "Real-Time Nucleic Acid Sequence-Based Amplification Is More Convenient than Real-Time PCR for Quantification of Plasmodium falciparum." Journal of Clinical Microbiology 43, no. 1 (January 1, 2005): 402–5. http://dx.doi.org/10.1128/jcm.43.1.402-405.2005.

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39

Dwiyitno, Dwiyitno, Stefan Hoffman, Koen Parmentier, and Chris Van Keer. "Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination." Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology 13, no. 3 (December 30, 2018): 115. http://dx.doi.org/10.15578/squalen.v13i3.370.

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Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

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40

Dellière, Sarah, Maud Gits-Muselli, P. Lewis White, Carlo Mengoli, Stéphane Bretagne, and Alexandre Alanio. "Quantification of Pneumocystis jirovecii: Cross-Platform Comparison of One qPCR Assay with Leading Platforms and Six Master Mixes." Journal of Fungi 6, no. 1 (December 26, 2019): 9. http://dx.doi.org/10.3390/jof6010009.

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Diagnosis of Pneumocystis jirovecii pneumonia relies on nucleic acid quantification in respiratory samples. Lack of standardization among molecular assays results in significant differences among assays/centers. To further promote standardization, we compared four thermocyclers and six master mixes for the detection of P. jirovecii. Whole nucleic acid (WNA) was extracted from broncho-alveolar lavages. Positive and negative sample extracts were pooled to get enough homogeneous materials. Three master mixes were tested to detect DNA by qPCR (D1, D2, and D3), and three to detect WNA by reverse transcriptase qPCR (W1, W2, and W3) manufactured by Roche, Eurogentec, Applied Biosystem, Invitrogen and Thermofischer Scientific. Experiments were performed on four thermocyclers (Roche LightCycler 480, Qiagen Rotor-Gene Q, Applied Biosystem ABI7500, and QuantStudio). Comparison of quantitative cycle (Cq) values between the methods targeting WNA versus DNA showed lower Cq values for WNA, independently of thermocycler and master mix. For high and low fungal loads, ∆Cq values between DNA and WNA amplification were 6.97 (±2.95) and 5.81 (±3.30), respectively (p < 0.0001). Regarding DNA detection, lower Cqs were obtained with D1 compared to D2 and D3, with median ∆Cq values of 2.6 (p = 0.015) and 2.9 (p = 0.039) respectively. Regarding WNA detection, no mix was superior to the others. PCR efficiency was not significantly different according to the qPCR platform (p = 0.14). This study confirmed the superiority of WNA over DNA detection. A calibration method (e.g., an international standard) for accurate comparative assessment of fungal load seems necessary.

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41

Li, Jin, Fengfei Wang, Harvey Mamon, Matthew H. Kulke, Lyndsay Harris, Elizabeth Maher, Lilin Wang, and G. Mike Makrigiorgos. "Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples." Clinical Chemistry 52, no. 4 (April 1, 2006): 624–33. http://dx.doi.org/10.1373/clinchem.2005.063321.

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Abstract Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide “tail” fluorescently labeled at the 5′ end. An oligonucleotide complementary to this tail, carrying a 3′ quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r2 >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

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42

Chen, Zhiwei, Nigel G. Halford, and Chenghong Liu. "Real-Time Quantitative PCR: Primer Design, Reference Gene Selection, Calculations and Statistics." Metabolites 13, no. 7 (June 28, 2023): 806. http://dx.doi.org/10.3390/metabo13070806.

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Real-time quantitative PCR is a technique that can measure the content of the target nucleic acid sequence of interest in a given sample. It is mainly divided into absolute and relative quantitative methods. The relative quantification is mainly used in gene expressions for functional genomic and transcriptome studies. However, to use this technology accurately, there are some key points to master. First, specific primers need to be designed to ensure amplification of the gene of interest (GOI). Second, the appropriate reference gene or reference gene combination has to be selected. Finally, scientific gene expression level calculations and statistics are required to obtain accurate results. Therefore, this work proposes a workflow for relative quantitative PCR and introduces the relevant points so that beginners can better understand and use this technology.

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43

Tan, Susanna K., Stephen Milligan, Malaya K. Sahoo, Nathaniel Taylor, and Benjamin A. Pinsky. "Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus." Journal of Clinical Microbiology 55, no. 3 (January 4, 2017): 923–30. http://dx.doi.org/10.1128/jcm.02315-16.

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ABSTRACT Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X − 0.28, R 2 = 0.99; secondary standard, Y = 1.04X − 0.26, R 2 = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, −0.52 to −1.01; IU/ml, 0.07 to −0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, −0.10 log 10 ; copies/ml, −0.70 log 10 ). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

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Ulrich, Robert M., Erica T. Casper, Lisa Campbell, Bill Richardson, Cynthia A. Heil, and John H. Paul. "Detection and quantification of Karenia mikimotoi using real-time nucleic acid sequence-based amplification with internal control RNA (IC-NASBA)." Harmful Algae 9, no. 1 (January 2010): 116–22. http://dx.doi.org/10.1016/j.hal.2009.08.010.

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45

TEH, HUEY-FANG, NAVEEN RAMALINGAM, HAI-QING GONG, and SWEE-NGIN TAN. "MICROFLUIDIC FLOW-THROUGH REACTOR WITH ELECTROCHEMICAL SENSOR ARRAY FOR REAL-TIME PCR." Modern Physics Letters B 23, no. 03 (January 30, 2009): 369–72. http://dx.doi.org/10.1142/s0217984909018424.

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We developed an integrated microfluidic flow-through EC-PCR (EC-PCR) microdevice for the concurrent DNA amplification, PCR products EC detection and PCR products quantification instead of the current available fluorescence detection scheme. The microfluidic flow-through EC-PCR microdevice was fabricated with the state-of-the-art microfabrication technology, by bonding a bottom glass substrate having a microelectrode array to a top glass cover having the microchannels made of PDMS material. Both the amplification of the target DNA sequence and the subsequent EC detection of the PCR products were carried out concurrently on the integrated device by real-time monitoring. The underlying principle of the microfluidic flow-through EC-PCR method was based on the changes of current signal of methylene blue (MB), which worked as an electrochemically active species DNA intercalator in the PCR mixture, during the amplification process at the extension phase. The results shown in this work indicated that the nucleic acid analysis could be performed in a fast thermal cycling and true real-time quantitative electrochemical detection. The signal variation trends of the EC detection and the fluorescence detection were the same in our verification measurements for both methods, which suggested that the EC detection method was feasible for this application.

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46

Murthy, Shweta, Akash Suresh, Debabrata Dandasena, Sakshi Singh, Madhusmita Subudhi, Vasundhra Bhandari, Vandna Bhanot, Jaspreet Singh Arora, and Paresh Sharma. "Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis." Pathogens 12, no. 2 (February 10, 2023): 296. http://dx.doi.org/10.3390/pathogens12020296.

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Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

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47

Schoepp, Nathan G., Travis S. Schlappi, Matthew S. Curtis, Slava S. Butkovich, Shelley Miller, Romney M. Humphries, and Rustem F. Ismagilov. "Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples." Science Translational Medicine 9, no. 410 (October 4, 2017): eaal3693. http://dx.doi.org/10.1126/scitranslmed.aal3693.

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Rapid antimicrobial susceptibility testing (AST) is urgently needed for informing treatment decisions and preventing the spread of antimicrobial resistance resulting from the misuse and overuse of antibiotics. To date, no phenotypic AST exists that can be performed within a single patient visit (30 min) directly from clinical samples. We show that AST results can be obtained by using digital nucleic acid quantification to measure the phenotypic response ofEscherichia colipresent within clinical urine samples exposed to an antibiotic for 15 min. We performed this rapid AST using our ultrafast (~7 min) digital real-time loop-mediated isothermal amplification (dLAMP) assay [area under the curve (AUC), 0.96] and compared the results to a commercial (~2 hours) digital polymerase chain reaction assay (AUC, 0.98). The rapid dLAMP assay can be used with SlipChip microfluidic devices to determine the phenotypic antibiotic susceptibility ofE. colidirectly from clinical urine samples in less than 30 min. With further development for additional pathogens, antibiotics, and sample types, rapid digital AST (dAST) could enable rapid clinical decision-making, improve management of infectious diseases, and facilitate antimicrobial stewardship.

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48

Tulloch, Rachel L., Karan Kim, Chisha Sikazwe, Alice Michie, Rebecca Burrell, Edward C. Holmes, Dominic E. Dwyer, Philip N. Britton, Jen Kok, and John-Sebastian Eden. "RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples." Viruses 15, no. 4 (April 19, 2023): 1006. http://dx.doi.org/10.3390/v15041006.

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Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.

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49

Taskin, Bilgin, Ayse Gul Gozen, and Metin Duran. "Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification." Applied and Environmental Microbiology 77, no. 13 (May 20, 2011): 4329–35. http://dx.doi.org/10.1128/aem.02895-10.

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ABSTRACTQuantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed withEscherichia coliATCC 25922 as the model organism and theuidAgene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

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Chen, L., B. Shopsin, Y. Zhao, D. Smyth, G. A. Wasserman, C. Fang, L. Liu, and B. N. Kreiswirth. "Real-Time Nucleic Acid Sequence-Based Amplification Assay for Rapid Detection and Quantification of agr Functionality in Clinical Staphylococcus aureus Isolates." Journal of Clinical Microbiology 50, no. 3 (January 4, 2012): 657–61. http://dx.doi.org/10.1128/jcm.06253-11.

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