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Laboratory verification of an RT-PCR assay for SARS-CoV-2.

ABSTRACT: BACKGROUND:Reverse transcription-polymerase chain reaction (RT-PCR) is an extremely common clinical method for detecting pathogens, particularly for emerging infectious diseases such as the new coronavirus disease (COVID-19). Currently, detection of the RNA from the novel coronavirus SARS-CoV-2 is the gold standard for establishing a COVID-19 diagnosis. This study evaluates the characteristic performance of the analytical system in a clinical laboratory. METHODS:A commercial SARS-CoV-2 RNA RT-PCR Kit used in a clinical laboratory is assessed based on ISO 15189 verification requirements. A multiple real-time RT-PCR assay for the RdRP, N, and E genes in SARS-CoV-2 is verified. RESULTS:The analytical system exhibits good analytical sensitivity (1000 copies/mL) and specificity (100%); however, the values of 86.7% and 100% for analytical accuracy deserved attention, compared with two other types of methods. Overall, the kit is potentially useful for SARS-CoV-2 diagnostic testing and meets the verification requirements. CONCLUSION:Compliance with international standards, such as ISO 15189, is valuable for clinical laboratories and for improving laboratory medicine quality and safety. Normalization is essential for obtaining reliable results from the SARS-CoV-2 RNA RT-PCR assay. This study aims to develop an improved SARS-CoV-2 verification framework compared with traditional molecular diagnostic methods, given the urgency of implementing new assays in clinical laboratories.

SUBMITTER: Wu Y

PROVIDER: S-EPMC7435359 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Laboratory verification of an RT-PCR assay for SARS-CoV-2.

Wu Yingping Y Xu Wei W Zhu Zhiqiang Z Xia Xiaoping X

Journal of clinical laboratory analysis 20200804 10

<h4>Background</h4>Reverse transcription-polymerase chain reaction (RT-PCR) is an extremely common clinical method for detecting pathogens, particularly for emerging infectious diseases such as the new coronavirus disease (COVID-19). Currently, detection of the RNA from the novel coronavirus SARS-CoV-2 is the gold standard for establishing a COVID-19 diagnosis. This study evaluates the characteristic performance of the analytical system in a clinical laboratory.<h4>Methods</h4>A commercial SARS- ...[more]

PMID: 32754967

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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.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness.ImportanceThis study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

Dataset Information

Laboratory verification of an RT-PCR assay for SARS-CoV-2.

Publications

Laboratory verification of an RT-PCR assay for SARS-CoV-2.

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