The Establishment And Application Of A High-throughput Detection Method For Coronavirus Based On Multiplex PCR-mass Spectrometry

Coronaviruses(CoVs),of the family Coronaviridae,are a large group of viruses which include viruses that can infect animals or people.According to the latest criteria of International Committee on Taxonomy of Viruses,CoVs are classified into four genera based on phylogeny,including Alphacoronavirus,Betacoronavirus,Deltacoronavirus and Gammacoronavirus.There are six human coronaviruses(HCoVs),HCoV-229E,HCoV-OC43,HCoV-NL63,HCoV-HKU1,severe acute respiratory syndrome coronavirus(SARS-CoV),and Middle East respiratory syndrome coronavirus(MERS-CoV)which belong to Alphacoronavirus and Betacoronavirus.In most cases,HCoVs cause mild to moderate upper-respiratory illness with symptoms including runny nose,cough,sore throat,and fever.However SARS-CoV emerging in China in 2003 and MERS-CoV first reported in the Middle East in 2012 cause severe severe acute respiratory syndrome,with a fatality rate close to 10%and 40%respectively.The emergence of SARS-CoV and MERS-CoV suggests that highly pathogenic HCoVs remain a key threat to human health.Because of the severity and communicability of diseases caused by HCoVs,rapid and sensitive diagnostic methods are essential for the timely implementation of effective infection control measures to prevent further transmission.The large number of tourists and pilgrims from across the globe traveling to the Middle East also increases the risk of transmitting MERS-CoV to other areas.Thus,it is important to develop a high-throughput and sensitive method that can monitor the distribution of HCoVs,especially MERS-CoV.In view of the lessons we learned from MERS and SARS outbreak,Co Vs have the ability of interspecies transmission and may emerge as novel pathogens.Therefore,an ideal diagnosis method should not only identify known HCoVs accurately,but also have the ability to provide clues for the emerging HCoVs.Our work aims to develop a sensitive method(mCoV-MS)which can not only identify known HCoVs accurately,but also have the ability to provide clues for the emerging HCoVs.The method was performed using multiplex PCR couple with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)system.We developed a 17-plex analysis to detect six HCoVs in Panel A and another 17-plex analysis to detect Alphacoronavirus and Betacoronavirus in Panel B.All tested primers and probes for the mCoV-MS method were effective,with no cross-reactivity observed with other common respiratory viruses.To confirm the usefulness of the mCoV-MS method we screened 384 pharyngeal and/or anal swab samples collected from bats/rodents,and 131 nasal and throat swabs from human patients.The results showed good concordance with the results of metagenomic analysis or PCR-sequencing.The validation test showed mCoV-MS method can detect potentially pathogenic CoVs in Alphacoronavirus and Betacoronavirus and provide convincingly phylogenetic evidences about unknown CoVs.The mCoV-MS method is a sensitive assay that is relatively simple to carry out.The validation test showed mCoV-MS method can detect potentially pathogenic CoVs in Alphacoronavirus and Betacoronavirus,which provides a powerful measure for dynamic monitoring of infection and prevalence of related coronavirus.We propose that this method be used to complement next generation sequencing technology for large-scale screening studies,in response to preventing and alerting emerging infectious diseases caused by novel coronavirus.The mCoV-MS method also provides a new model for surveillance of emerging infectious diseases.