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1
Carrino, John J., and Helen H. Lee. "Nucleic acid amplification methods." Journal of Microbiological Methods 23, no. 1 (1995): 3–20. http://dx.doi.org/10.1016/0167-7012(95)00024-f.
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Birkenmeyer, Larry G., and Isa K. Mushahwar. "DNA probe amplification methods." Journal of Virological Methods 35, no. 2 (1991): 117–26. http://dx.doi.org/10.1016/0166-0934(91)90127-l.
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Bonilla, Luis Fabián, Jamison H. Steidl, Grant T. Lindley, Alexei G. Tumarkin, and Ralph J. Archuleta. "Site amplification in the San Fernando Valley, California: Variability of site-effect estimation using the S-wave, coda, and H/V methods." Bulletin of the Seismological Society of America 87, no. 3 (1997): 710–30. http://dx.doi.org/10.1785/bssa0870030710.
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Abstract During the months that followed the 17 January 1994 M 6.7 Northridge, California, earthquake, portable digital seismic stations were deployed in the San Fernando basin to record aftershock data and estimate site-amplification factors. This study analyzes data, recorded on 31 three-component stations, from 38 aftershocks ranging from M 3.0 to M 5.1, and depths from 0.2 to 19 km. Site responses from the 31 stations are estimated from coda waves, S waves, and ratios of horizontal to vertical (H/V) recordings. For the coda and the S waves, site response is estimated using both direct spectral ratios and a generalized inversion scheme. Results from the inversions indicate that the effect of Qs can be significant, especially at high frequencies. Site amplifications estimated from the coda of the vertical and horizontal components can be significantly different from each other, depending on the choice of the reference site. The difference is reduced when an average of six rock sites is used as a reference site. In addition, when using this multi-reference site, the coda amplification from rock sites is usually within a factor of 2 of the amplification determined from the direct spectral ratios and the inversion of the S waves. However, for nonrock sites, the coda amplification can be larger by a factor of 2 or more when compared with the amplification estimated from the direct spectral ratios and the inversion of the S waves. The H/V method for estimating site response is found to extract the same predominant peaks as the direct spectral ratio and the inversion methods. The amplifications determined from the H/V method are, however, different from the amplifications determined from the other methods. Finally, the stations were grouped into classes based on two different classifications, general geology and a more detailed classification using a quaternary geology map for the Los Angeles and San Fernando areas. Average site-response estimates using the site characterization based on the detailed geology show better correlation between amplification and surface geology than the general geology classification.
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Parshikov, B. Y., N. V. Prudnikov, E. A. Leonova, and N. A. Solovyov. "Femtosecond Laser Pulses Amplification Methods." Russian Journal of General Chemistry 94, no. 9 (2024): 2538–44. http://dx.doi.org/10.1134/s1070363224090317.
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Altwegg, M., and J. Verhoef. "Amplification methods in diagnostic microbiology." Journal of Microbiological Methods 23, no. 1 (1995): 1–2. http://dx.doi.org/10.1016/0167-7012(95)00020-l.
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Bodulev, O. L., and A. M. Soloviev. "HIGHLY SENSITIVE METHODS FOR MICRORNA DETECTION USING ISOTHERMAL METHODS FOR NUCLEIC ACIDS AMPLIFICATION." http://eng.biomos.ru/conference/articles.htm 1, no. 19 (2021): 244–46. http://dx.doi.org/10.37747/2312-640x-2021-19-244-246.
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This work will present the results of the development of two new highly sensitive heterogeneous methods for the determination of miRNA-141 and miRNA-39 using the isothermal circular strand-displacement amplification and the catalytic hairpin assembly methods for analyte amplification.
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Park, Jee-Woong. "Principles and Applications of Loop-Mediated Isothermal Amplification to Point-of-Care Tests." Biosensors 12, no. 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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Padzil, Faiz, Abdul Razak Mariatulqabtiah, Wen Siang Tan, et al. "Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research." Animals 12, no. 1 (2021): 76. http://dx.doi.org/10.3390/ani12010076.
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Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.
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Zanoli, Laura Maria, and Giuseppe Spoto. "Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices." Biosensors 3 (December 27, 2012): 18–43. https://doi.org/10.3390/bios3010018.
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Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.
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Wolcott, M. J. "Advances in nucleic acid-based detection methods." Clinical Microbiology Reviews 5, no. 4 (1992): 370–86. http://dx.doi.org/10.1128/cmr.5.4.370.
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Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.
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Zulkharnain, Azham, Norhafizah Sidek, and Awang Ahmad Sallehin Husaini. "Evaluation of different methods for total DNA extraction from sago pith residue." Journal of Biochemistry, Microbiology and Biotechnology 2, no. 2 (2014): 67–69. http://dx.doi.org/10.54987/jobimb.v2i2.150.
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Direct extraction of DNA from the environment has become major importance for molecular analyses for the study of microbial communities in soil and other decomposing agrowaste. The presence of humic substances in Sago pith residue not only results in low DNA quality but also can lead to PCR amplification inhibition which may hinder most molecular studies. Many of the published protocols have been found to be unsuitable to obtained high amount of yield and low of humic acids contamination. This study presents the evaluation of three different methods for extracting total DNA from Sago pith residue. The methods evaluated were enzymatic lysis, glass bead homogenization and freeze-thaw treatment. Each method were evaluated by 260/280 nm absorbance ratio for protein contamination, 260/230 nm absorbance ratio for other contaminants and PCR amplification for molecular work suitability. Among the three methods, freeze-thaw treatment provided the highest yield of DNA, 5.06±0.01 μg/g of Sago pith residue. Nevertheless, all three methods resulted in poor DNA quality which could be used for PCR amplification. Additional steps of agarose electrophoresis and silica column purification were found to be effective for increasing the quality of the extracted DNA and were validated by positive PCR amplifications.
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Buhot, Arnaud. "Advances in Amplification Methods for Biosensors." Biosensors 13, no. 3 (2023): 365. http://dx.doi.org/10.3390/bios13030365.
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Today, there is a rapidly growing demand for sensitive and selective biosensors in various domains, including environmental monitoring such as (waste)water control, detection of pollution for personal/public safety, agricultural/food safety and quality control, veterinary and medical diagnostics, etc [...]
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Martín, Maria P., and Katarina Winka. "Alternative Methods of extracting and Amplifying Dna from lichens." Lichenologist 32, no. 2 (2000): 189–96. http://dx.doi.org/10.1006/lich.1999.0254.
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AbstractWe have investigated whether DNA extraction protocols designed specifically for fungi and/or lichens perform better on lichens than do corresponding protocols designed for plants and insects. Two different PCR-amplification protocols were used to evaluate the quality of the DNA extracted with each method. The DNA extractions with highest quality were obtained with the protocols designed for insects and plants, and the most successful amplifications were obtained with Ready-To-Go PCR Beads. This indicates that fungal or lichen specific protocols might not be necessary for successful extraction of high quality DNA from lichens.
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Moťková, P., and J. Vytřasová. "Comparison of methods for isolating fungal DNA." Czech Journal of Food Sciences 29, Special Issue (2012): S76—S85. http://dx.doi.org/10.17221/266/2011-cjfs.
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In this study methods of fungal DNA isolation were optimised and compared. The aim of the isolation processes was to obtain DNA of sufficient quality and quantity necessary for its amplification, as most detection techniques require DNA amplification before the proper DNA detection itself. For this purpose, classic methods of DNA extraction were compared and optimised while isolations using commercial kits were also done. The methods were evaluated from several perspectives, with focus especially laid on the isolated DNA not contain PCR inhibitors which would prevent DNA amplification, thus inhibiting the detection itself. For optimising the individual methods, collection strains of the genus Aspergillus were used. After the evaluation, two most suitable methods were selected and chosen for isolating potentially aflatoxigenic moulds taken from food samples. These methods were the commercially supplied kit for isolating DNA from plant leaves from Sigma and a classic method according Cenis in combination with the cell wall disruption by means of liquid nitrogen.
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Yüce, Meral, Naimat Ullah, and Hikmet Budak. "Trends in aptamer selection methods and applications." Analyst 140, no. 16 (2015): 5379–99. http://dx.doi.org/10.1039/c5an00954e.
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Aptamers are target specific ssDNA, RNA or peptide sequences generated by anin vitroselection and amplification method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment), which involves repetitive cycles of binding, recovery and amplification steps.
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Ivanov, Aleksandr V., Irina V. Safenkova, Natalia V. Drenova, Anatoly V. Zherdev, and Boris B. Dzantiev. "Comparison of Biosensing Methods Based on Different Isothermal Amplification Strategies: A Case Study with Erwinia amylovora." Biosensors 12, no. 12 (2022): 1174. http://dx.doi.org/10.3390/bios12121174.
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Isothermal amplifications allow for the highly sensitive detection of nucleic acids, bypassing the use of instrumental thermal cycling. This work aimed to carry out an experimental comparison of the four most promising techniques: recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) coupled with lateral flow test or coupled with additional amplification based on CRISPR/Cas12a resulting from the fluorescence of the Cas12a-cleaved probe. To compare the four amplification techniques, we chose the bacterial phytopathogen Erwinia amylovora (causative agent of fire blight), which has a quarantine significance in many countries and possesses a serious threat to agriculture. Three genes were chosen as the targets and primers were selected for each one (two for RPA and six for LAMP). They were functionalized by labels (biotin, fluorescein) at the 5′ ends for amplicons recognition by LFT. As a result, we developed LAMP-LFT, LAMP-CRISPR/Cas, RPA-LFT, and RPA-CRISPR/Cas for E. amylovora detection. The detection limit was 104 CFU/mL for LAMP-LFT, 103 CFU/mL for LAMP-CRISPR/Cas, and 102 CFU/mL for RPA-LFT and RPA-CRISPR/Cas. The results of four developed test systems were verified by qPCR on a panel of real samples. The developed assays based on RPA, LAMP, CRISPR/Cas12a, and LFT are rapid (30–55 min), user-friendly, and highly sensitive for E. amylovora detection. All proposed detection methods can be applied to fire blight diagnosis and effective management of this disease.
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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 (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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Erber, Ramona, Matthias Rübner, Simon Davenport, et al. "Impact of fibroblast growth factor receptor 1 (FGFR1) amplification on the prognosis of breast cancer patients." Breast Cancer Research and Treatment 184, no. 2 (2020): 311–24. http://dx.doi.org/10.1007/s10549-020-05865-2.
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Abstract Purpose Various aberrations in the fibroblast growth factor receptor genes FGFR1, FGFR2, and FGFR3 are found in different cancers, including breast cancer (BC). This study analyzed the impact of FGFR amplification on the BC prognosis. Methods The study included 894 BC patients. The amplification rates of FGFR1, FGFR2, and FGFR3 were evaluated on tissue microarrays using fluorescence in situ hybridization (FISH). Associations between these parameters and prognosis were analyzed using multivariate Cox regression analyses. Results FGFR1 FISH was assessable in 503 samples, FGFR2 FISH in 447, and FGFR3 FISH in 562. The FGFR1 amplification rate was 6.6% (n = 33). Increased FGFR2 copy numbers were seen in 0.9% (n = 4); only one patient had FGFR3 amplification (0.2%). Most patients with FGFR1 amplification had luminal B-like tumors (69.7%, n = 23); only 32.6% (n = 153) of patients without FGFR1 amplification had luminal B-like BC. Other patient and tumor characteristics appeared similar between these two groups. Observed outcome differences between BC patients with and without FGFR1 amplification did not achieve statistical significance; however, there was a trend toward poorer distant metastasis-free survival in BC patients with FGFR1 amplification (HR = 2.08; 95% CI 0.98 to 4.39, P = 0.05). Conclusion FGFR1 amplification occurs most frequently in patients with luminal B-like BC. The study showed a nonsignificant correlation with the prognosis, probably due to the small sample size. Further research is therefore needed to address the role of FGFR1 amplifications in early BC patients. FGFR2 and FGFR3 amplifications are rare in patients with primary BC.
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Wei, Zhigang, Jie Wang, Hua Bai, et al. "FGFR1 amplification and EGFR mutation in Chinese squamous cell lung cancer." Journal of Clinical Oncology 30, no. 15_suppl (2012): 7545. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.7545.
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7545 Background: Squamous cell lung cancer (SCC) lacks for effective targeted therapies. FGFR1 amplification has emerged as a potential biomarker. This study aimed to explore clinicopathologic characteristics of FGFR1 amplification in Chinese SCC patients and further explore the correlation between FGFR1 amplification and EGFR mutations. Methods: One hundred seventy-seven SCC patients were included in this retrospective study. Gene copy number of FGFR1 and EGFR mutations were detected by fluorescence in situ hybridization (FISH) and denaturing high-performance liquid chromatography (DHPLC), respectively. Results: FGFR1 amplifications were detected in 24.9% (44/177) Chinese SCC patients. FGFR1 amplification in SCC was more common in male (28.0%, 40/143) and smokers (28.7%, 39/136) than female (11.8%, 4/34, p=0.049) and nonsmokers (12.2%, 5/41, p=0.032). FGFR1 amplification and EGFR mutations were mutually exclusive (p=0.006), fourty-one of 139(29.4%) patients with wild-type EGFR had FGFR1 amplification, while 3 of 38 (7.9%) patients with EGFR mutation had FGFR1 amplification. Conclusions: FGFR1 amplification was common in Chinese squamous cell lung cancer, and mutually exclusive with EGFR mutations.
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Silva, Arnaldo Neves Santos, Jordy Coffa, Yohei Miyagi, et al. "Coamplification of receptor tyrosine kinases and downstream targets in Japanese gastric cancers." Journal of Clinical Oncology 32, no. 3_suppl (2014): 41. http://dx.doi.org/10.1200/jco.2014.32.3_suppl.41.
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41 Background: Amplifications of receptor tyrosine kinases genes (RTKs), EGFR, ERBB2, FGFR2, MET, have been associated with the pathogenesis and progression of gastric cancer (GC). Recent studies have found that coamplifications of RTKs are rare in GC. Two phase III trials using RTK targeted therapy have failed to achieve survival benefit in GC patients. Co-amplification of RTKs and downstream targets genes (DSTs), PIK3CA, KRAS, MYC, CCNE1 may be related to resistance to RTK targeted therapy. We tested the hypothesis whether RTKs and DSTs genes are coamplified in GC. Methods: DNA and RNA were extracted from 221 GC from the Kanagawa Cancer Center Hospital (Yokohama, Japan). Copy number of EGFR, ERBB2, FGFR2, PIK3CA, KRAS, MYC, CCNE1 was investigated by a newly developed multiplex ligation dependent probe amplification (MLPA) assay. RNA expression of the same genes was measured using the NanoString platform and compared to DNA copy number status. The frequency of RTK and DST co-amplifications was established and related to KRAS mutation status. Results: RTK amplification was associated with high levels of RNA expression except MYC (all p<0.05). 68% of GC had no RTK amplification. 9% of GC had RTK amplification and no DST amplification. 10% of GC had RTKs co-amplified. 23% of GC had RTK and DST co-amplified. There was no overlap between KRAS mutations and amplifications of KRAS, PIK3CA, FGFR2 or MET. Conclusions: This is the first study reporting that RTK and DST co-amplification are more frequent than RTK co-amplification in GC. These results indicate that GC patients considered for RTK targeting therapy might require DNA copy number assessment of RTKs as well as key DSTs and KRAS mutation testing in order to identify those patients who benefit most from treatment.
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Hubbard, Roger A. "Human Papillomavirus Testing Methods." Archives of Pathology & Laboratory Medicine 127, no. 8 (2003): 940–45. http://dx.doi.org/10.5858/2003-127-940-hptm.
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Abstract Testing for human papillomavirus (HPV) relies exclusively on techniques of molecular biology using nucleic acid probes. Tests for HPV using nucleic acid probes have been commercially available since the late 1980s, but early tests were cumbersome, involving the use of nucleic acid probes labeled with radioactive phosphorus (32P). These early HPV tests did not achieve widespread use because they did not detect all oncogenic HPV genotypes. The current commercial HPV detection kit, Digene's Hybrid Capture 2 kit, detects virtually all high-risk oncogenic HPV types, as well as most low-risk nononcogenic HPV genotypes. The Hybrid Capture 2 test format is a proprietary nucleic acid hybridization signal amplification system owned by Digene Corporation. Virtually all test formats for DNA sequence analysis are amenable to applications intended to detect and perhaps quantify the various HPV genotypes. These methods can involve direct hybridization with complementary DNA probes, such as Southern blotting or in situ hybridization, signal amplification, such as the Hybrid Capture 2 method or target nucleic acid amplification, most notably the polymerase chain reaction (PCR). Polymerase chain reaction has been used for HPV detection, genotyping, and viral load determination. General or consensus primer–mediated PCR assays have enabled screening for a broad spectrum of HPV types in clinical specimens using a single PCR reaction. Following amplification using consensus primers, individual HPV genotypes are identified using a variety of methods. Using consensus primers in a test format known as real-time quantitative PCR (RQ-PCR), it is possible to generate viral load (concentration) data from reaction curves generated by monitoring PCR reaction kinetics in real time.
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Olkhov-Mitsel, Ekaterina, Danny Chan, Kenneth J. Craddock, et al. "Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting ERBB2 and Other Gene Amplifications in Solid Tumors." Cancers 16, no. 23 (2024): 3927. http://dx.doi.org/10.3390/cancers16233927.
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Background: Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. Methods: This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. Results: We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10–30%, 31–60%, and 61–95% groups revealed significantly higher gene amplification detection rates in the 31–60% and 61–95% groups versus the 10–30% group (20.6% and 26.7% vs. 9.2%, p < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10–30% group versus the 61–95% group (0.11 vs. 0.52; p < 0.0001). To further investigate the analytic performance of OCAv3 in detecting ERBB2 amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed ERBB2 status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. Conclusions: This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.
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Kluth, M., M. Hitzschke, M. Lennartz, et al. "MYC amplifications are a common event in urothelial bladder carcinomas associated with an aggressive tumor phenotype." American Journal of Clinical Pathology 160, Supplement_1 (2023): S91—S92. http://dx.doi.org/10.1093/ajcp/aqad150.202.
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Abstract Introduction/Objective Gene amplifications of the proto-oncogen MYC are common events in carcinomas. In this study, we analyzed the impact of MYC amplifications on tumor aggressiveness and patient prognosis in urothelial bladder carcinomas. Methods/Case Report MYC copy number status was analyzed on more than 2,500 urothelial bladder carcinomas in a tissue microarray format by using dual-labeling fluorescence in-situ hybridization (FISH) with probes for centromere 8 and MYC (8q24). The results were compared with tumor phenotype and patient outcome. Results (if a Case Study enter NA) MYC amplification (ratio MYC/centromere 8 ≥2) occurred in 10% of 2,052 analyzable urothelial bladder carcinomas. Of these, 141 (6.8%) had a low level MYC copy number gain (MYC/centromere 8 ratio ≥2 and ≤ 3) and 68 (3.3%) had high-level MYC amplifications (MYC/centromere 8 ratio &gt;3) . The rate of MYC gain and MYC amplification increased from pTa G2 low (0.6%), to pTa G2 high (7.7%), and pTa G3 (8.2%) carcinomas, and were highest in muscle-invasive pT2-4 (13.2%) carcinomas (p&lt;0.0001). In muscle-invasive urothelial carcinomas, the MYC amplification rate increased from pT2 (13.1%), to pT3 (14.7%), and pT4 (19.8%) but these differences failed to reach statistical significance (p=0.0902). MYC amplification was also unrelated to patient survival in 526 patients with pT2-4 urothelial carcinomas who had undergone cystectomy (p=0.5526). Conclusion MYC amplification is a common event in urothelial bladder carcinoma - especially in case of pTa G3 and muscle-invasive carcinoma. The association of MYC amplification with advanced stage may reflect increasing genomic instability going along with tumor progression.
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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 (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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Peano, Clelia, Marco Severgnini, Ingrid Cifola, Gianluca De Bellis, and Cristina Battaglia. "Transcriptome amplification methods in gene expression profiling." Expert Review of Molecular Diagnostics 6, no. 3 (2006): 465–80. http://dx.doi.org/10.1586/14737159.6.3.465.
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Skaradzińska, Aneta, Marta Ochocka, Paulina Śliwka, et al. "Bacteriophage amplification – A comparison of selected methods." Journal of Virological Methods 282 (August 2020): 113856. http://dx.doi.org/10.1016/j.jviromet.2020.113856.
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Germann, Daniel, and Amalio Telenti. "Nucleic acid amplification methods in diagnostic virology." Journal of Microbiological Methods 23, no. 1 (1995): 31–39. http://dx.doi.org/10.1016/0167-7012(95)00022-d.
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Karch, Helge, Andreas Schwarzkopf, and Herbert Schmidt. "Amplification methods in diagnostic bacteriology (selected examples)." Journal of Microbiological Methods 23, no. 1 (1995): 55–73. http://dx.doi.org/10.1016/0167-7012(95)00026-h.
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Salem K, Alketbi. "Collection Methods for Touch DNA Direct Amplification." Forensic, Legal & Investigative Sciences 9, no. 1 (2023): 1–4. http://dx.doi.org/10.24966/flis-733x/100072.
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Jorayev, Nabijon, Lobar Akbarova, Gulasal Ozodova, and Rahimjon Kholmamatov. "Modern diagnostic methods of chlamydia." Multidisciplinary Journal of Science and Technology 5, no. 4 (2025): 1013–19. https://doi.org/10.5281/zenodo.15288340.
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Modern diagnostic methods for chlamydia, particularly Chlamydia trachomatis, have evolved significantly and include several approaches. Here are some of the most commonly used methods: Nucleic Acid Amplification Tests (NAATs), PCR (Polymerase Chain Reaction), LAMP (Loop-mediated Isothermal Amplification), Swab Tests, Serological Tests, Rapid Tests,Point-of-Care Testing,Combination Testing. The organization of modern diagnostic methods for chlamydia is a multifaceted approach that balances accuracy, accessibility, speed, cost, and patient-centered care. By considering these factors, healthcare providers can effectively diagnose and manage chlamydia infections, ultimately contributing to better sexual health outcomes in the population.
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Chemisova, O. S., O. A. Tsyrulina, A. L. Trukhachev, and A. K. Noskov. "Comparative analysis of methods for isothermal amplification of nucleic acids." Journal of microbiology, epidemiology and immunobiology 99, no. 1 (2022): 126–38. http://dx.doi.org/10.36233/0372-9311-176.
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In this review, methods for isothermal amplification of nucleic acids are considered and analyzed, in particular, loop isothermal amplification of DNA and RNA (LAMP/RT-LAMP), helicase-dependent amplification (HDA) and recombinase polymerase amplification (RPA). The advantages and disadvantages of each of the techniques are described. The possibility of their application in the molecular diagnostics of infectious diseases is evaluated. A brief review of the literature on the use of LAMP, HDA, RPA in the diagnostics of viral, bacterial infections and diseases of protozoal etiology was conducted. It has been shown that the LAMP method has a number of advantages over other diagnostic methods: high efficiency, specificity, simplicity, turnaround time and minimum requirements for instrument equipment. As a result, it is concluded that loop isothermal amplification is a promising method for detecting the DNA/RNA of various pathogens. The data on the introduction of the LAMP method in the diagnostics of particularly dangerous bacterial and viral infections, including for the detection of RNA of a new coronavirus infection (SARS-CoV-2) in clinical samples, are presented.
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Jorayev, Nabijon, Lobar Akbarova, Gulasal Ozodova, and Rahimjon Kholmamatov. "Modern diagnostic methods of chlamydia." Multidisciplinary Journal of Science and Technology 5, no. 4 (2025): 1056–62. https://doi.org/10.5281/zenodo.15307585.
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Modern diagnostic methods for chlamydia, particularly Chlamydia trachomatis, have evolved significantly and include several approaches. Here are some of the most commonly used methods: Nucleic Acid Amplification Tests (NAATs), PCR (Polymerase Chain Reaction), LAMP (Loop-mediated Isothermal Amplification), Swab Tests, Serological Tests, Rapid Tests,Point-of-Care Testing,Combination Testing. The organization of modern diagnostic methods for chlamydia is a multifaceted approach that balances accuracy, accessibility, speed, cost, and patient-centered care. By considering these factors, healthcare providers can effectively diagnose and manage chlamydia infections, ultimately contributing to better sexual health outcomes in the population.
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Zhang, Min, Jiajia Wu, Zhaoai Shi, et al. "Molecular Methods for Identification and Quantification of Foodborne Pathogens." Molecules 27, no. 23 (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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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 (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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Şafak, Erdal. "Models and Methods to Characterize Site Amplification from a Pair of Records." Earthquake Spectra 13, no. 1 (1997): 97–129. http://dx.doi.org/10.1193/1.1585934.
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The paper presents a tutorial review of the models and methods that are used to characterize site amplification from the pairs of rock- and soil-site records, and introduces some new techniques with better theoretical foundations. The models and methods discussed include spectral and cross-spectral ratios, spectral ratios for downhole records, response spectral ratios, constant amplification factors, parametric models, physical models, and time-varying filters. An extensive analytical and numerical error analysis of spectral and cross-spectral ratios shows that probabilistically cross-spectral ratios give more reliable estimates of site amplification. Spectral ratios should not be used to determine site amplification from downhole-surface recording pairs because of the feedback in the downhole sensor. Response spectral ratios are appropriate for low frequencies, but overestimate the amplification at high frequencies. The best method to be used depends on how much precision is required in the estimates.
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Lee, Kwang Ho, Seong Hoon Jeong, Seung Woo Han, and Kang Su Kim. "The Effect of Seismic Design Methods on Design Eccentricities in Building with Planar Irregularities." Applied Mechanics and Materials 764-765 (May 2015): 1149–53. http://dx.doi.org/10.4028/www.scientific.net/amm.764-765.1149.
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Seismic provisions have utilized design eccentricities to reduce planar irregularities in lateral stiffness of buildings. In calculating a design eccentricity, the dynamic amplification factor may be applied either to accidental eccentricity or to both inherent and accidental eccentricities according to design codes. In this paper, different code provisions and their impact on torsional responses of buildings are investigated using example buildings with various aspect ratios and eccentricities. It was found that dynamic amplification is underestimated if the inherent eccentricity is small, when buildings are designed by seismic provisions using dynamic amplification factors for both to inherent and accidental eccentricities. On the other hand, the design eccentricity determined by applying the dynamic amplification factor only to accidental eccentricity reflects torsional amplification accurately.
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Steinwald, Peter, Elisa Ledet, Bryce Raymon Christensen, et al. "Cell-free DNA (cfDNA) analysis and evaluation of BRAF amplifications and mutations in metastatic castration-resistant prostate cancer." Journal of Clinical Oncology 36, no. 6_suppl (2018): 255. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.255.
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255 Background: Cell-free DNA (cfDNA) is an accessible method for characterizing tumoral alterations. We report cfDNA screenings of prostate cancer pts positive for BRAF amplifications/mutations in pts with metastatic CRPC. Methods: Guardant360 testing (Guardant Health, Inc.) assesses cell-free DNA analysis using sequencing to identify genomic alterations in 73 cancer-related genes in circulation. A total of 133 metastatic castrate resistant prostate cancer (mCRPC) pts in various stages of therapy had Guardant cfDNA analyses. Treatment histories prior to testing and concurrent cfDNA alterations were analyzed. Results: BRAF amplifications were detected in 32 (24%) mCRPC pts; 5 pts had concurrent BRAF mutations. Of the mutations detected, only one (K601E, n = 2) was a known activating mutation while all others were variants of unknown significance (VUS). One K601E mutation pt had no other cfDNA alterations. Additionally, 4 pts without BRAF amplification had VUS BRAF mutations. BRAF amplification pts had ≥ 2 concurrent gene amplifications/alterations with the median being 8. The most common recurrent amplifications/alterations were AR (75%), p53 (59%), CDK6 (53%), MET (50%), and MYC (50%). Abiraterone (Abi) and/or Enzalutamide (Enza) resistance was associated with BRAF amplification (p = 0.0042). Non-Abi/Enza resistance pts were less likely to have BRAF amplification. The 2 pts with BRAF K601E mutation were treated with targeted protocol therapy without success however one K601E pts was subsequently treated with cabazitaxel+carboplatin which produced a positive clinical response and a 99.79% reduction in PSA. Conclusions: Pts resistant to Abi/Enza have an increased risk of developing BRAF amplifications. BRAF amplifications arise in the context of multiple additional detectable cfDNA alterations. Identification of actionable mutations, such as BRAF K601E, illustrates the potential for cfDNA testing to direct pt treatment. As cfDNA profiling continues to expand, the ability translate alterations into clinically actionable strategies is critical.
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Dunbar, W. Scott, and Robin G. Charlwood. "Empirical Methods for the Prediction of Response Spectra." Earthquake Spectra 7, no. 3 (1991): 333–53. http://dx.doi.org/10.1193/1.1585632.
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Three empirical methods of estimating response spectra are reviewed. It is shown that these methods produce comparable results, provided that the comparisons are made in a consistent manner. Although the traditional method of scaling amplification factors by peak ground motions is fundamentally reasonable, there exist some significant discrepancies between scaled and other types of spectra. The basic reason for this is that the amplification factors commonly used in the engineering community are biased toward those of a large magnitude earthquake recorded on a soil site. The dependence of spectral shape on magnitude and site conditions can be accounted for by independent estimates of peak ground acceleration, velocity and displacement to scale an unbiased set of amplification factors derived for particular site conditions. Other methods of estimating spectra account for the dependence of spectral shape on magnitude and site conditions in a more direct manner. In order to reduce the discrepancies between scaled and other types of spectra, it is recommended that a new set of amplification factors be estimated using a suite of response spectra due to earthquakes whose magnitudes are evenly distributed in the range 5.0 ≤ M ≤ 7.5 and which have been recorded on sites having similar soil conditions.
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Kim, Kyoung-Ho, and Jin-Woo Bae. "Amplification Methods Bias Metagenomic Libraries of Uncultured Single-Stranded and Double-Stranded DNA Viruses." Applied and Environmental Microbiology 77, no. 21 (2011): 7663–68. http://dx.doi.org/10.1128/aem.00289-11.
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ABSTRACTInvestigation of viruses in the environment often requires the amplification of viral DNA before sequencing of viral metagenomes. In this study, two of the most widely used amplification methods, the linker amplified shotgun library (LASL) and multiple displacement amplification (MDA) methods, were applied to a sample from the seawater surface. Viral DNA was extracted from viruses concentrated by tangential flow filtration and amplified by these two methods. 454 pyrosequencing was used to read the metagenomic sequences from different libraries. The resulting taxonomic classifications of the viruses, their functional assignments, and assembly patterns differed substantially depending on the amplification method. Only double-stranded DNA viruses were retrieved from the LASL, whereas most sequences in the MDA library were from single-stranded DNA viruses, and double-stranded DNA viral sequences were minorities. Thus, the two amplification methods reveal different aspects of viral diversity.
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Raghav, Kanwal Pratap Singh, Chad Tang, M. Pia Morelli, et al. "Multiple independent methods fail to confirm MET amplification rate reported in literature for metastatic colorectal cancer (mCRC)." Journal of Clinical Oncology 33, no. 3_suppl (2015): 572. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.572.
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572 Background: MET inhibition is emerging as a potent therapeutic strategy and MET gene amplification has shown predictive significance. MET amplification rate in mCRC, as previously reported in literature, varies from 9% in primary to 18% in metastases but intermixes increased copy number from chromosomal level aberrations with focal gene amplification. Validation of MET amplification rate in mCRC is needed. Methods: We performed analyses of MET amplification in mCRC patients (pts) (n = 636) across multiple cohorts. Cohort 1 (n = 103) included tissue microarray from liver metastases analysed using fluorescence in situ hybridization (FISH) [cMET and CEP7 probes, MET/CEP7 ratio > 2]. Cohort 2 (n = 205) included pts referred for phase I trials who had MET amplification testing using FISH. Cohort 3 (n = 279) included cases sequenced with HiSeq (Illumina) with full exome coverage for 202 genes including MET (average depth 800) with focal gene amplification (≥ 4 copies) identified by an in-house algorithm. Cohort 4 (n = 49) included pts refractory to EGFR monoclonal antibodies enrolled in the ATTACC (a prospective molecular screening) program for mCRC, in whom plasma circulating-free DNA (cfDNA) was analyzed by Guardant sequencing technology. Results: In tissue based analyses, focal MET amplification rate was 1.7% and was higher in primary tumors compared to metastases [3.1% (9/291) vs. 0.4% (1/288), p = 0.02] [Table]. In cohorts 2 & 3 MET amplification was found in 4 [MET/CEP7: 2.1 – 7.7; primary (4/130), metastases (0/75)] and 6 [copy number: 4.0 – 6.7; primary (5/161), metastases (1/110)] cases, respectively. MET amplification rate in pts who had progressed on anti-EGFR therapy was 14.3% (Table). Conclusions: Contrary to prior reports, in this large cohort, MET amplification was a rare event in mCRC pts and the rate was not higher in metastatic sites. However, MET amplification occurred in a sizable subset of pts refractory to anti-EGFR therapy as identified by cfDNA analysis. MET amplification appears to play a minor role in de novo colorectal carcinogenesis but may play an important role in acquired resistance to anti-EGFR therapy. [Table: see text]
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Yoon, Harry H., Qian Shi, William R. Sukov, et al. "HER2 testing in esophageal adenocarcinoma (EAC) using parallel tissue-based methods." Journal of Clinical Oncology 31, no. 4_suppl (2013): 2. http://dx.doi.org/10.1200/jco.2013.31.4_suppl.2.
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2 Background: Testing for HER2 in EAC has become routine given its ability to predict benefit from HER2-targeted therapy. HER2 protein analysis by immunohistochemistry (IHC) is more rapid and generally less expensive than assessment of gene amplification by fluorescence in situ hybridization (FISH). However, the accuracy of IHC for predicting HER2 amplification has not been examined in a large EAC cohort. Methods: Surgical EAC specimens were examined from 675 patients who underwent curative resection at a single institution without preoperative therapy. Tumors were located in the esophagus (47%) or gastroesophageal junction (53%); most were node-positive (73%). Every tumor was evaluated by IHC in parallel with FISH in a blinded manner using FDA-approved assays. A consensus IHC score was determined by 2 pathologists using tumor-specific criteria (negative, 0 or 1+; equivocal, 2+; positive, 3+). Gene amplification by FISH was defined as a HER2/CEP17ratio ≥ 2. Results: HER2 amplification was detected in 89% of IHC 3+ cases, 13% of IHC 2+ cases, and 4% of IHC 0-1+ cases (Table). Accordingly, using FISH as the reference standard, the positive predictive value (PPV) of a positive IHC test (3+) was 89% (79/89 [95% CI 82%-95%]), and the negative predictive value of a negative IHC test (0-1+) was 96% (401/417 [94%-98%]). Importantly, the PPV of an equivocal IHC score (2+) for detecting HER2 amplification was 13% (22/169 [8%-18%]). Conclusions: In the largest study to date comparing HER2 testing methods in EAC, a negative IHC result (0 or 1+) nearly excludes the presence of gene amplification by FISH. Whereas a positive IHC result (3+) strongly predicts for the presence of amplification, an equivocal IHC result (2+) is a weak predictor. These findings in EAC support a HER2 testing algorithm where IHC is used for initial screening and FISH testing is restricted to cases with equivocal IHC results. [Table: see text]
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Mahony, James B. "Detection of Respiratory Viruses by Molecular Methods." Clinical Microbiology Reviews 21, no. 4 (2008): 716–47. http://dx.doi.org/10.1128/cmr.00037-07.
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SUMMARY Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.
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Gullett, Jonathan C., and Frederick S. Nolte. "Quantitative Nucleic Acid Amplification Methods for Viral Infections." Clinical Chemistry 61, no. 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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Chen, Hui-Ling, Meng-Meng Guo, Hao Tang, et al. "Nucleic acid amplification-based methods for microRNA detection." Analytical Methods 7, no. 6 (2015): 2258–63. http://dx.doi.org/10.1039/c4ay02938k.
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Swaminathan, Bala, and Timothy J. Barrett. "Amplification methods for epidemiologic investigations of infectious diseases." Journal of Microbiological Methods 23, no. 1 (1995): 129–39. http://dx.doi.org/10.1016/0167-7012(95)00021-c.
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Björnerfeldt, Susanne, and Carles Vilà. "Evaluation of methods for single hair DNA amplification." Conservation Genetics 8, no. 4 (2006): 977–81. http://dx.doi.org/10.1007/s10592-006-9220-z.
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Klümper, Niklas, Ngoc Khanh Tran, Stefanie Zschaebitz, et al. "Occurrence of NECTIN4 amplification in solid tumors and enfortumab vedotin response in metastatic urothelial cancer." Journal of Clinical Oncology 42, no. 4_suppl (2024): 673. http://dx.doi.org/10.1200/jco.2024.42.4_suppl.673.
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673 Background: The anti-NECTIN4 antibody-drug conjugate (ADC) enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains are frequent genomic events in mUC leading to NECTIN4 amplifications. Here, we aimed to evaluate the potential of NECTIN4 amplification as a genomic biomarker to predict EV response in patients with mUC. Methods: We established a NECTIN4-specific fluorescence in-situ hybridization (FISH) assay to assess NECTIN4 copy number variations (CNVs) in a multicenter EV-treated mUC patient cohort (UC-EV, N=77), and CNV data were correlated with membranous NECTIN4 protein expression (H-score) assessed via immunohistochemistry, EV treatment responses and outcomes. Next, we conducted a pan-cancer analysis of the The Cancer Genome Atlas (TCGA) datasets, comprising 10,712 patients across 32 cancer types, to investigate the relationship between NECTIN4 CNV, mRNA expression (RNAseq) and protein levels (RPPA) across entities. Results: In TCGA cohorts, NECTIN4 amplification occurs frequently across different solid cancer types, especially in 15-20% of bladder cancers (17% in TCGA-BLCA), as well as 5-10% in breast cancer (TCGA-BRCA) and lung adenocarcinoma (TCGA-LUAD). We confirmed the amplification frequency in our UC-EV cohort (18%). NECTIN4 amplification is significantly associated with both increased NECTIN4 mRNA expression (e.g., TCGA-BLCA, BRCA, LUAD) and membranous NECTIN4 protein expression (UC-EV), and represents a stable genomic alteration during metastatic progression. In the UC-EV cohort, all patients with NECTIN4 amplification (N=14) responded to EV. In multivariable Cox adjusted for age and sex, NECTIN4 amplifications led to a 93% risk reduction for death compared to a NECTIN4non-amplified status (HR=0.07, 95%-CI 0.01–0.53; P<0.001). Conclusions: Our study highlights the value of NECTIN4 amplifications to predict EV responses in patients with mUC. NECTIN4 amplifications occur frequently in different cancer types and therefore have the potential to be a novel tumor-agnostic genomic biomarker that enables tailored NECTIN4-targeted therapies in various entities.
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Nai, Yi Heng, Egan H. Doeven, and Rosanne M. Guijt. "An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein." PLOS ONE 17, no. 3 (2022): e0265391. http://dx.doi.org/10.1371/journal.pone.0265391.
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The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.
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Kato, Shumei, Jeffrey S. Ross, Laurie Gay, et al. "Analysis of MDM2 Amplification: Next-Generation Sequencing of Patients With Diverse Malignancies." JCO Precision Oncology, no. 2 (November 2018): 1–14. http://dx.doi.org/10.1200/po.17.00235.
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Purpose MDM2 amplification can promote tumorigenesis directly or indirectly through p53 inhibition. MDM2 has increasing clinical relevance because inhibitors are under evaluation in clinical trials, and MDM2 amplification is a possible genomic correlate of accelerated progression, known as hyperprogression, after anti–PD-1/PD-L1 immunotherapy. We used next-generation sequencing (NGS) to ascertain MDM2 amplification status across a large number of diverse cancers. Methods We interrogated the molecular profiles of 102,878 patients with diverse malignancies for MDM2 amplification and co-altered genes using clinical-grade NGS (182 to 465 genes). Results MDM2 amplification occurred in 3.5% of patients (3,650 of 102,878). The majority of tumor types had a small subset of patients with MDM2 amplification. Most of these patients (99.0% [3,613/3,650]) had co-alterations that accompanied MDM2 amplification. Various pathways, including those related to tyrosine kinase (37.9% [1,385 of 3,650]), PI3K signaling (25.4% [926 of 3,650]), TP53 (24.9% [910 of 3,650]), and MAPK signaling (23.6% [863 of 3,650]), were involved. Although infrequent, mismatch repair genes and PD-L1 amplification also were co-altered (2.2% [79 of 3,650]). Most patients (97.6% [3,563 of 3,650]) had one or more co-alterations potentially targetable with either a Food and Drug Administration–approved or investigational agent. MDM2 amplifications were less frequently associated with high tumor mutation burden compared with the MDM2 wild-type population (2.9% v 6.5%; P < .001). An illustrative patient who harbored MDM2 amplification and experienced hyperprogression with an immune checkpoint inhibitor is presented. Conclusion MDM2 amplification was found in 3.5% of 102,878 patients, 97.6% of whom harbored genomic co-alterations that were potentially targetable. This study suggests that a small subset of most tumor types have MDM2 amplification as well as pharmacologically tractable co-alterations.
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Yu, Jieli, Minghui Wang, RongFeng Song, et al. "The landscape of chromosome 11q13 amplification in Chinese solid tumor patients and hyperprogressive disease (HPD) clinical example." Journal of Clinical Oncology 38, no. 15_suppl (2020): e15222-e15222. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15222.
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e15222 Background: Multiple biomarkers are thought to be effective guides in selecting immune checkpoint inhibitors, such as Tumor mutational burden (TMB), PD-L1 and MSI/dMMR. Immunotherapy may be miraculous effective in some patients but many other patients experienced poor prognosis and even tumor overgrowth after immunotherapy in real practice. Previous studies have reported that 11q13 (CCND1, FGF3, FGF4, and FGF19) amplifications were associated with HPD. However, the characterization of chromosome 11q13 amplification in Chinese solid tumor patients are not clear. Methods: A total of 10167 Chinese solid tumor patients’ FFPE and matching blood samples sequenced by next-generation sequencing (NGS) targeting 450 cancer genes were included in this study. Genomic alterations including single nucleotide variants (SNV), insertions and deletions, copy number variations and fusions were assessed. The testing was carried out by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Results: Chromosome 11q13 amplifications were identified in 679 (6.7%) Chinese solid tumor patients. The top six tumor types with 11q13 amplification were esophageal carcinoma 43.8%, melanoma 17%, urothelial carcinoma 16%, breast carcinoma 13.4%, head and neck carcinoma 13.1% and hepatocellular carcinoma 9.4%. In 2257 patients with TMB-H (10muts/Mb) / PD-L1 expression / MSI samples, chromosome 11q13 copy number gain were identified in 184 (8.2%) patients. Chromosome 11q13 amplification and MSI were independent events and didn't occur simultaneously. In TMB-H patients, the detection rate of 11q13 amplification was 8.5%, while in PD-L1 positive patients, the amplification rate of 11q13 was 7.4%. In patients with TMB-H and PD-L1 positive, the detection rate of 11q13 amplification was 9.4%. In our clinical practice, we saw example of HPD exist in a lung squamous cell carcinoma patient who was treated with multiple lines of therapy and then used the Nivolumab. The patient underwent genetic testing and was found to be chromosome 11q13 amplified. Conclusions: The proportion of 11q13 amplification was different in different types of solid tumor. Esophageal carcinoma, melanoma and urothelial carcinoma were top three types of solid tumors with 11q13 amplification. The 11q13 amplification did not coincide with MSI but it overlapped with PD-L1 expression or TMB-H. Together, these results may provide immunotherapy guidance in clinical practice.