High sensitivity detection of coronavirus SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method

Chenyu Li; David N. Debruyne; Julia Spencer; Vidushi Kapoor; Lily Y. Liu; Bo Zhou; Lucie Lee; Rounak Feigelman; Grayson Burdon; Jeffrey Liu; Alejandra Oliva; Adam Borcherding; Hongdong Tan; Alexander E. Urban; Guoying Liu; Zhitong Liu

A severe epidemic coronavirus (SARS-CoV-2) infection, now just characterized as a pandemic by the World Health Organization (WHO), started in December of 2019 in Wuhan China and quickly spread to many countries in the world [1] [2] [3] . It has caused over 7,000 deaths as of mid-March and made daily impacts on various aspects of societal life around the globe 4 . SARS-CoV-2 is a coronavirus with positive-sense, singlestranded RNA about 30kb in length. The genome of SARS-CoV-2 is currently under careful investigation [5] [6] [7] [8] [9] and being extensively modeled according to the Chinese National Center for Biological information (2019 New Coronavirus Information Database, https://bigd.big.ac.cn/ncov). Of note, SARS-CoV-2 exhibits over 99% sequence similarity among many sequenced isolates, and is also highly similar to other coronaviruses 5,9 . Present methods for detecting SARS-CoV-2 have been reported and discussed 10, 11 , including RT-PCR, serological testing 12 and reverse transcription-loop-mediated isothermal amplification 13, 14 . Currently, RT-PCR is considered the gold standard of diagnosis for SARS-CoV-2 due to its ease of use and high sensitivity. RT-PCR has been reported to detect SARS-CoV-2 in saliva 15 , pharyngeal swab, blood, anal swab 16 , urine, stool 17 , and sputum specimens 18 . In laboratory conditions, the RT-PCR methodology has been shown to detect 4-8 copies of virus, through amplification of targets in the Orf1ab, E and N viral genes, at 95% confidence intervals [19] [20] [21] . However, only about 47-59% of the positive cases were identified by RT-PCR, and 75% of RT-PCR negative results were actually found to be positive, with repeated tests required 17, [22] [23] [24] . In addition, there is evidence suggesting that heat inactivation of clinical samples causes loss of virus particles, thereby hindering the efficiency of downstream diagnosis 25 .