Comparison and Sensitivity Evaluation of Three Different Commercial Real-Time Quantitative PCR Kits for SARS-CoV-2 Detection

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the “gold standard” diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen’s κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.

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Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density

Animals ◽

10.3390/ani11061605 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1605

Author(s):

Annika Wichert ◽

Esra Einax ◽

Natalie Hahn ◽

Anne Klassen ◽

Karsten Donat

Keyword(s):

Real Time ◽

Mycobacterium Avium ◽

Detection Probability ◽

Mycobacterium Avium Subspecies Paratuberculosis ◽

Positive Association ◽

Significant Loss ◽

Positive Sample ◽

Bacterial Density ◽

Fecal Samples ◽

Real Time Quantitative Pcr

Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

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One-step RNA extraction for RT-qPCR detection of 2019-nCoV

10.1101/2020.04.02.022384 ◽

2020 ◽

Cited By ~ 5

Author(s):

Monica Sentmanat ◽

Evguenia Kouranova ◽

Xiaoxia Cui

Keyword(s):

Real Time ◽

Rna Extraction ◽

Virus Transmission ◽

Extraction Methods ◽

Viral Rna ◽

Taqman Probe ◽

N Gene ◽

Structural Protein ◽

Diagnostic Panel ◽

Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

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Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.023887-0 ◽

2011 ◽

Vol 60 (4) ◽

pp. 508-514 ◽

Cited By ~ 10

Author(s):

Olivia Peuchant ◽

Jean Philippe Duvert ◽

Maïthé Clerc ◽

Sophie Raherison ◽

Christiane Bébéar ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Real Time ◽

Dna Quantification ◽

Mrna Transcription ◽

Real Time Quantitative Pcr ◽

Dna And Rna ◽

Rna Quantification ◽

Additional Cell

The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

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Comparison of In-House Real-Time Quantitative PCR to the Adenovirus R-Gene Kit for Determination of Adenovirus Load in Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00976-10 ◽

2010 ◽

Vol 48 (9) ◽

pp. 3132-3137 ◽

Cited By ~ 25

Author(s):

H. Jeulin ◽

A. Salmon ◽

P. Bordigoni ◽

V. Venard

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Clinical Samples ◽

R Gene ◽

Real Time Quantitative Pcr

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Determination of RhD Zygosity: Comparison of a Double Amplification Refractory Mutation System Approach and a Multiplex Real-Time Quantitative PCR Approach

Clinical Chemistry ◽

10.1093/clinchem/47.4.667 ◽

2001 ◽

Vol 47 (4) ◽

pp. 667-672 ◽

Cited By ~ 89

Author(s):

Rossa W K Chiu ◽

Michael F Murphy ◽

Carrie Fidler ◽

Benny C Y Zee ◽

James S Wainscoat ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Gene Dosage ◽

Amplification Refractory Mutation System ◽

Pcr Assay ◽

Real Time Quantitative Pcr ◽

Mutation System ◽

Allele Specific ◽

Quantitative Pcr Assay

Abstract Background: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. Methods: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, ΔCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. Results: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the ΔCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. Conclusion: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.

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Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

Journal of Medical Microbiology ◽

10.1099/jmm.0.010587-0 ◽

2009 ◽

Vol 58 (7) ◽

pp. 878-883 ◽

Cited By ~ 11

Author(s):

Wafa Habbal ◽

Fawza Monem ◽

Barbara C. Gärtner

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Isolates ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Pcr Product ◽

Highly Sensitive ◽

Primer Sets ◽

Analytical Sensitivities ◽

Strain Ad169

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

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