Analytical sensitivity and efficiency comparisons of SARS-COV-2 qRT-PCR assays
By
Chantal BF Vogels,
Anderson F Brito,
Anne L. Wyllie,
Joseph R Fauver,
Isabel M. Ott,
Chaney C Kalinich,
Mary E. Petrone,
Arnau Cassanovas-Massana,
M. Catherine Muenker,
Adam J. Moore,
Jonathan Klein,
Peiwen Lu,
Alice Lu-Culligan,
Xiaodong Jiang,
Daniel J. Kim,
Eriko Kudo,
Tianyang Mao,
Miyu Moriyama,
Ji Eun Oh,
Annsea Park,
Julio Silva,
Eric Song,
Takehiro Takehashi,
Manabu Taura,
Maria Tokuyama,
Arvind Venkataraman,
Orr-El Weizman,
Patrick Wong,
Yexin Yang,
Nagarjuna R Cheemarla,
Elizabeth White,
Sarah Lapidus,
Rebecca Earnest,
Bertie Geng,
Pavithra Vijayakumar,
Camila Odio,
John Fournier,
Santos Bermejo,
Shelli Farhadian,
Charles Dela Cruz,
Akiko Iwasaki,
Albert Ko,
Marie-Louise Landry,
Ellen F. Foxman,
Nathan D. Grubaugh
Posted 01 Apr 2020
medRxiv DOI: 10.1101/2020.03.30.20048108
The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined "N1" and "N2" assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.
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