Investigating the optimum sample type and target genes for SARS-CoV-2 detection

Aims The cycle threshold (Ct) value for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection is important because of the criteria for quarantine management, including release from it, which are defined in Guidelines on the Novel Coronavirus-Infected Pneumonia Diagnosis and Treatment (Provisional 9th Edition, China). As this is also currently relevant because of the recent SARS-CoV-2 epidemic in Shanghai, we discuss the SARS-CoV-2 nucleic acid detection and its problems. We focus on the gene fragments and sample types involved in nucleic acid detection and their effect on the latest criteria for release from quarantine. Methods A total of 215 patients with SARS-CoV-2 infection were included. Pharyngeal swabs (nasopharyngeal swabs plus oropharyngeal swabs) were collected in the early stage of the disease, and pharyngeal swabs, sputum samples, and anal swabs were collected both in the middle and advanced stages of the disease. The Open reading frame 1ab (ORF lab) gene, Nucleocapsid (N) gene and Envelop (E) gene of each sample were quantitatively analyzed using fluorescence qPCR technique. Results Exclusion of the E gene detection results had no significant effect on the interpretation of the nucleic acid Ct value of 35, with a positive concordance rate of 98.7% (95% CI 86.0%–100%) and an overall concordance rate of 99.7% (95% CI 92.9%–100%). The kappa coefficient was 0.99 (95% CI 0.92–1.00). Compared with nucleic acid detection using both pharyngeal swab and sputum sample, the positive concordance rate of the detection using pharyngeal swab alone was 47.6% (95% CI 27.8%–99.3%). The kappa coefficient was 0.63 (95% CI 0.53–0.75), and the consistency was not ideal. Conclusions Nucleic acid detection using the ORF 1ab gene and the N gene can achieve the purpose of SARS-CoV-2 detection. Nucleic acid detection using sputum samples is significant in the determination of Ct values and its significance in the development of the criteria for release from quarantine needs to be taken into account. It is suggested that to increase the accuracy of nucleic acid detection, instead of unilaterally pursuing increasing the number of target genes for amplification and improving PCR techniques, more attention should be paid to sampling and sample reliability, as well as strict quality control of the detection process. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The study was funded by the Changzheng Hospital Fund (2019CZJS210-2) and ( 2020YLCYJ-Y03) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The trial was approved by the ethics committee of Changzheng Hospital. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * COVID-19 : coronavirus disease 2019 Ct : cycle threshold PCR : polymerase chain reaction SARS-CoV-2 : severe acute respiratory syndrome coronavirus 2 ORF lab gene : open reading frame 1ab gene N gene : Nucleocapsid gene E gene : Envelop gene