Prevalence and genetic diversity analysis of human coronaviruses among cross-border children

Liu, Peilin; Shi, Lei; Zhang, Wei; He, Jianan; Liu, Chunxiao; Zhao, Chunzhong; Kong, Siu Kai; Loo, Jacky Fong Chuen; Gu, Dayong; Hu, Longfei

Viral nucleic acids were extracted from 200 μL respiratory samples using MagNA pure 96 DNA with Viral NA small volume kit (Roche) and EZ1 virus Mini kit V2.0 (Qiagen) according to the manufacturer's instructions. The viral nucleic acids were stored at −80°C until use. For the coronaviruses screening, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed in triplicate using ABI 7500 qRT-PCR thermocycler. The specimens were firstly screened for influenza viruses according to the procedure previously published [17] . Samples of negative results on influenza were then tested for pan-coronavirus as well as 13 other common respiratory viruses. The qRT-PCR master mixture was performed according to the manufacturer's instructions of qRT-PCR Kit (Quant), mainly contained 20.0 μL buffer and 5.0 μL RNA. The thermal cycling conditions were set as follows: reverse transcription at 50°C for 10 min, initial 95°C for 3 min, 40 cycles of PCR amplification at 95°C for 15 s, annealing/elongation at 60°C for 45 s. The partial S (S1 subunit) and RdRp genes were detected in the positive samples after HCoVs screening with the forward (F) and reverse (R) primers listed in Table 1 . The PCR mixture (25 μL) contained 5.0 μL of RNA, PCR buffer mixed with Superscript ®III/PT Taq Kit (Invitrogen) containing 12.5 μl of 2× Rxn Mix,1 μL of forward and reverse primer (10 μM), 1.0 μL of MgSO 4 , 1.0 μL of BSA (0.1%),1.0 μl of Superscript ®III/PT Taq Enzyme, 0.5 μL of RNA Inhibitor, 2.0 μL of nuclease free water. The thermal cycling conditions were set as follows: reverse transcription at 50°C for 30 min, 35 cycles of PCR amplification at 94°C for 30 s, annealing at 50-54°C for 30 s, elongation at 68°C for 150-180 s, final elongation at 68°C for 5 min. Sanger sequencing (Sangon Biotech) of the PCR products of concentration ranging from 50 to 300 ng/μL was performed to study the homology and mutations of samples. Genetic sequence data have been submitted to a publicly available repository (Genbank) and the accessible sequence accession numbers (MF996589-MF996664) including features of the samples and sequences. (Fig. 1a) . The results of the clinical symptoms of these samples were shown in Table 2 .