scholarly journals Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit

Critical Care ◽  
2010 ◽  
Vol 14 (4) ◽  
pp. R159 ◽  
Author(s):  
Katsunori Yanagihara ◽  
Yuko Kitagawa ◽  
Masao Tomonaga ◽  
Kunihiro Tsukasaki ◽  
Shigeru Kohno ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Dna Detection ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Pathogen Dna

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

scholarly journals Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting

2020 ◽  
Author(s):  
Fabiola Mancini ◽  
Fabrizio Barbanti ◽  
Maria Scaturro ◽  
Stefano Fontana ◽  
Angela Di Martino ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Reverse Transcription ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Activity Period ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. Methods A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2–infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. Results The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. Conclusions This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

scholarly journals Evaluation of Amplification Targets for the Specific Detection ofBordetella pertussisUsing Real-Time Polymerase Chain Reaction

2014 ◽  
Vol 25 (4) ◽  
pp. 217-221 ◽  
Author(s):  
Mohammad Rubayet Hasan ◽  
Rusung Tan ◽  
Ghada N Al-Rawahi ◽  
Eva Thomas ◽  
Peter Tilley
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Target Genes ◽  
Reference Panel ◽  
Superior Performance ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

scholarly journals Simultaneous One-Tube Quantification of Host and Pathogen DNA with Real-Time Polymerase Chain Reaction

2002 ◽  
Vol 92 (1) ◽  
pp. 112-116 ◽  
Author(s):  
L. M. Winton ◽  
J. K. Stone ◽  
L. S. Watrud ◽  
E. M. Hansen
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Fluorescent Dyes ◽  
Quantitative Polymerase Chain Reaction ◽  
Positive Control ◽  
Douglas Fir ◽  
Chain Reaction ◽  
Taqman Probes ◽  
Polymerase Chain ◽  
Pathogen Dna

Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were β-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.

scholarly journals Systematic Review and Meta-analysis of Real-time Polymerase Chain Reaction assay for the detection of COVID-19 from clinical samples in low-and middle-income countries: Protocol

2021 ◽  
Author(s):  
Emmanuel Oladipo Babafemi
Keyword(s):  
Systematic Review ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meta Analysis ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Middle Income ◽  
Polymerase Chain

Abstract Background: COVID-19 has spread globally since its discovery in Hubei province, China in December 2019 and became pandemic in 2020. COVID-19 is a new betacoronavirus and a variant of severe acute respiratory syndrome coronavirus 2 (SARA- CoV-2). Rapid, accurate and reliable diagnosis of COVID-19 will prevent the spread and allow for appropriate management. The main objective of this systematic review is to identify, appraise and summarise the published evidence on the diagnostic performance and effectiveness of SARS-CoV-2 virus in the diagnosis of current or previous COVID-19 using real-time polymerase chain reaction (RT-PCR) assay in low-and middle-income countries (LMICs). Methods: We will search MEDLINE/PubMed, EMBASE, BIOSIS, LILACS, Cochrane Infectious Diseases Group Specialised Register (CIDG SR), Global Health, and CINAHL for published studies for the diagnosis of COVID-19 using real-time polymerase chain reaction assay in LMICs There will be no restriction regarding the language, date of publication, and publication status. We will include retrospective, cross-sectional and cohort observational studies will be included in the review. Selection of studies, data extraction and management, assessment of risk of bias, and quality of evidence will be performed by two independent reviewers (EB and BC). A third researcher (GM) will be consulted in case of discrepancies. Depending on the availability and quality of the data, a meta-analysis will be performed. Otherwise, findings will be qualitatively reported. Discussion: To our knowledge, this is the first systematic review and meta-analysis to assess the uptake of RT-PCR assay for SARS-CoV-2 detection from clinical samples in human in LMICs. This review will make available evidence on the uptake, accuracy, approach, and interpretation of results of this assay in the context of COVID-19 diagnosis which will meet an urgent need, considering the diagnostic challenges of RT-PCR assay for COVID-19 diagnosis in humans. Systematic review registration: PROSPERO CRD42021271894

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30%-60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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