Abstract
BackgroundThe fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction.ResultsOptimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection.ConclusionWe successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.
Highlights
In December 2019, the world witnessed an unknown coronavirus emerging in Wuhan, China, first called 2019 novel coronavirus (2019-nCoV), and severe acuteFreppel et al Virol J (2020) 17:196 and accurate diagnostic tools of SARS-CoV-2 in order to isolate infected people quickly to reduce transmission
Our data show that direct use of undiluted Universal Transport Media (UTM) without RNA extraction interferes with the Real-time reverse transcription polymerase chain reaction (rRT-PCR), since only 50% (15/30) were detected positive (Fig. 1a)
From sample sets 1 and 2, we evaluated the impacts of specimen dilution and thermal lysis on detection rates of direct rRT-PCR compared to manual RNA extraction as a standard reference
Summary
In December 2019, the world witnessed an unknown coronavirus emerging in Wuhan, China, first called 2019 novel coronavirus (2019-nCoV), and severe acuteFreppel et al Virol J (2020) 17:196 and accurate diagnostic tools of SARS-CoV-2 in order to isolate infected people quickly to reduce transmission. Our concern was that specimen itself or transport media such as UTM would interfere with rRT-PCR efficiency. UTM is a stable viral transport medium allowing collection and transport of viruses and bacteria that contains protective proteins and antimicrobials, which could interfere with rRT-PCR enzymes. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene AllplexTM 2019-nCoV assay without RNA extraction
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