ZENA SARS-CoV-2 Direct Detection Kit

Abstract

The ongoing COVID-19 pandemic has sparked an unprecedented need for rapid diagnostic tests. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal swab (PN) followed by a chain reaction of the Quantitative reverse transcription-polymerase (RT-qPCR) to detect purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a serious bottleneck in COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that skips the RNA extraction step entirely, and we tested this hypothesis in a series of blinded clinical samples.

The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) that were shown to be positive for SARS-CoV-2 RNA by traditional clinical RT-qPCR diagnosis that included an RNA extraction. Therefore, direct RT-qPCR could be a first-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but with a result negative initial. This strategy would dramatically alleviate COVID-19 test supply hotspots and should be applicable worldwide.

Specimens

Nasopharyngeal and oropharyngeal swab.

Content

1 x 1.9 mL COVID-19 FAM, ROX, and human RNase P HEX multiplex qPCR master mix
1 x 0.1 ml RT Enzyme Mix
1 x 0.05 ml of COVID-19 virus control
1 x 1 ml PCR grade water (nuclease-free)

Methods

  1. Samples

University of Vermont: Clinical nasopharyngeal swabs were collected in 3 ml of M6 transport medium. The Vermont Department of Health confirmed that patients were negative (3) or positive (6) for COVID-19 using the CDC 2019-Novel Coronavirus (2019-nCoV) 1 Real-time RT-PCR Diagnostic Panel. Limited amounts of this material were available, so we initially collected equal sample amounts from two COVID-19 patients. Subsequently, four additional COVID-19 confirmed patients and four COVID-19 negative donors were individually evaluated for the presence of SARS-CoV-2 RNA or RNase P by direct RT-qPCR of the sample material.

University of Washington: Clinical nasopharyngeal swabs were collected in 3 ml of the universal transport medium (UTM). 150 patient samples were confirmed as COVID-19 positive by the University of Washington Medical Laboratory with ranges of viral loads (CT values ​​of SARS-CoV-2 with primer N2 from the original clinical test run at UW vary from 10.17 to 38.13) and a sufficient volume was selected for this study.

All patient samples at each test site were stored at 4 ° C between sample collection and transport to the laboratory, and again until clinical testing was carried out, maintaining a standardized sample acquisition and processing protocol.

2. RNA extraction

University of Vermont: To compare the effect of nucleic acid extraction with direct RT-PCR, RNA was extracted from a pooled patient sample using the QIAamp Viral RNA Mini kit (Qiagen, Cat. No. 52904) according to the manufacturer’s instructions. . 140 µL of pooled nasopharyngeal swab diluent was removed and eluted in 60 µL; 5 µL (equivalent to 11 µL of the original sample) were used for real-time RT-PCR.

University of Washington: To compare the effect of nucleic acid extraction, 30 µL or 200 µL RNA was extracted from the patient sample using the Roche MagNA Pure 96 platform (Roche Lifesciences), eluted in 50 µL of buffer; 5 µL of RNA (equivalent to 3 µL of original sample for a 30 µL extraction) was used for real-time RT-PCR. To monitor RNA recovery and RT-PCR efficiency, 40,000 copies of EXO internal control RNA were added to the lysis buffer and subjected to the extraction process with each sample.

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