Enhancement of PCR efficiency using mono- and disaccharides

We fabricated a NOA81 PCR chip (NP-chip) and first proposed a simple BSA coating method to successfully overcome the low PCR efficiency induced by the inhibition of the NP-chip which seriously impacted its application in the area of gene amplification.

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Comparative evaluation of nested PCR for diagnosis of human brucellosis

10.21203/rs.2.16740/v1 ◽

2019 ◽

Author(s):

leila rahbarnia ◽

Safar Farajnia ◽

Behrooz Naghili ◽

Nazli Saeedi

Keyword(s):

Blood Culture ◽

Nested Pcr ◽

High Performance ◽

Human Brucellosis ◽

High Morbidity ◽

Chronic Infections ◽

Serological Tests ◽

Pcr Efficiency ◽

Sat 1 ◽

Serum Agglutination

Abstract Objectives: Brucellosis is a worldwide zoonotic disease with high morbidity in the absence of treatment. The early diagnosis of brucellosis is efficient to prevent chronic infections. The aim of this study is evaluation of nested PCR efficiency in comparison with conventional methods for diagnosis of human brucellosis. A total of 120 patients with brucellosis symptoms were included in this study. Serological and microbiological tests and nested PCR were used for detection of Brucella bacteria. Results: Based on serological tests, 60.83% (73/120) of individuals were positive for brucellosis which only 8.33% of cases were confirmed by blood culture. Among them, 55% of cases were positive in serum agglutination test (SAT≥1:160) and Coombs (C-SAT≥1:160) tests. Furthermore, 7 negative SAT cases were positive in C-SAT as evidence for chronic brucellosis. Also, 68.18% and 56.06% of SAT positive samples were positive in blood nested PCR and serum nested PCR respectively. The sensitivity of blood nested PCR was more than serum nested PCR, SAT≥1:160 and blood culture (P<0.001). The specificity of the blood and serum nested PCR was 100% compared with blood culture and SAT≥ 1:160. Our findings highlight high performance of nested PCR for diagnosis of both acute and chronic brucellosis.

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Comparison of commercial RT-PCR diagnostic kits for COVID-19

10.1101/2020.04.22.056747 ◽

2020 ◽

Cited By ~ 3

Author(s):

Puck B. van Kasteren ◽

Bas van der Veer ◽

Sharon van den Brink ◽

Lisa Wijsman ◽

Jørgen de Jonge ◽

...

Keyword(s):

Limit Of Detection ◽

Cross Reactivity ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Specific Method ◽

Preliminary Evaluation ◽

Rt Pcr ◽

Accurate Identification ◽

Clinical Sensitivity ◽

Pcr Efficiency

ABSTRACTThe final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed.The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene).We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity.PCR efficiency was ≥96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene.We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.

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Variation in PCR Efficiencies between Quantification Standards and Clinical Specimens using Different Real-Time Quantitative PCR Interpretation Methods

International Journal of Biology and Biomedical Engineering ◽

10.46300/91011.2020.14.6 ◽

2020 ◽

Vol 14 ◽

Keyword(s):

Real Time ◽

Clinical Samples ◽

Test Results ◽

Chain Reaction ◽

Established Method ◽

Standard Curve ◽

Real Time Quantitative Pcr ◽

Pcr Efficiency ◽

Polymerase Chain ◽

Inhibitory Agents

The analysis of the Ct and standard curve produced by real-time polymerase chain reaction (PCR) is a well-established method for the quantification of nucleic acids. However, this method assumes that the PCR efficiency between the unknown specimen and standard is equal, resulting in the possibility of significant inaccuracies due to the presence of inhibitory agents in the unknown specimen. Although numerous methods have been proposed to correct this issue, the understanding of the differences in PCR efficiencies in clinical samples is limited. In this study, 1185 cytomegalovirus (CMV) DNA real-time PCR test results from 106 batches were analyzed. The PCR efficiencies were calculated using the cpD2, maxE, Cy0, maxRatio and window-of-linearity (WoL) methods. The concentrations were calculated using the cpD2, Cy0, maxRatio, WoL, and take off point (TOP) methods. The coefficient of variation (CV) in the efficiency of the quantification standards was less than 5% in all methods. Positive samples with high quantification values demonstrated lower PCR efficiency compared to the quantification standards. This suggests possible inaccuracies in quantification using quantification standards in clinical samples.

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