Dataset Information

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

ABSTRACT:

Background

The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19.

Objective

We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2.

Methodology

Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real-time RT-PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.

Results

The limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of C_t values observed between two tests with r² = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.

Conclusion

In the present study, we established and validated an in-house real-time RT-PCR for molecular detection of SARS-CoV-2 in a resource-limited country.

SUBMITTER: Nguyen LT

PROVIDER: S-EPMC9102516 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Publications

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

Nguyen Linh Tung LT Nguyen Phuong Minh PM Dinh Duc Viet DV Pham Hung Ngoc HN Bui Lan Anh Thi LAT Vo Cuong Viet CV Nguyen Ben Huu BH Bui Hoan Duy HD Hoang Cuong Xuan CX Ngo Nhat Minh Van NMV Dang Truong Tien TT Do Anh Ngoc AN Vu Dung Dinh DD Nguyen Linh Thuy LT Nguyen Mai Ngoc MN Dinh Thu Hang Thi THT Ho Son Anh SA Hoang Luong Van LV Hoang Su Xuan SX Do Quyet Q

Journal of clinical laboratory analysis 20220321 5

<h4>Background</h4>The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19.<h4>Objective</h4>We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2.<h4>Methodology</h4>Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved ...[more]

PMID: 35312118

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Project description:BackgroundCOVID-19 is an ongoing public health pandemic regardless of the countless efforts made by various actors. Quality diagnostic tests are important for early detection and control. Notably, several commercially available one step RT-PCR based assays have been recommended by the WHO. Yet, their analytic and diagnostic performances have not been well documented in resource-limited settings. Hence, this study aimed to evaluate the diagnostic sensitivities and specificities of three commercially available one step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in Ethiopia in clinical setting.MethodsA cross-sectional study was conducted from April to June, 2021 on 279 respiratory swabs originating from community surveillance, contact cases and suspect cases. RNA was extracted using manual extraction method. Master-mix preparation, amplification and result interpretation was done as per the respective manufacturer. Agreements between RT-PCRs were analyzed using kappa values. Bayesian latent class models (BLCM) were fitted to obtain reliable estimates of diagnostic sensitivities, specificities of the three assays and prevalence in the absence of a true gold standard.ResultsAmong the 279 respiratory samples, 50(18%), 59(21.2%), and 69(24.7%) were tested positive by TIB, Da An, and BGI assays, respectively. Moderate to substantial level of agreement was reported among the three assays with kappa value between 0 .55 and 0.72. Based on the BLCM relatively high specificities (95% CI) of 0.991(0.973-1.000), 0.961(0.930-0.991) and 0.916(0.875-0.952) and considerably lower sensitivities with 0.813(0.658-0.938), 0.836(0.712-0.940) and 0.810(0.687-0.920) for TIB MOLBIOL, Da An and BGI respectively were found.ConclusionsWhile all the three RT-PCR assays displayed comparable sensitivities, the specificities of TIB MOLBIOL and Da An were considerably higher than BGI. These results help adjust the apparent prevalence determined by the three RT-PCRs and thus support public health decisions in resource limited settings and consider alternatives as per their prioritization matrix.

Project description:Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.

Dataset Information

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

Background

Objective

Methodology

Results

Conclusion

Publications

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

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