Detection of WN-Human1 sequence from clinical specimen. Naganori Nao, Kazuya Shirato, Shutoku Matsuyama, and Makoto Takeda Laboratory of Acute Viral Respiratory Infections and Cytokines, Department of Virology III, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, 208-0011 Tokyo, Japan
Method & Results Total RNA was extracted from four nasopharyngeal swabs using QIAamp viral RNA mini kit (Qiagen) following manufacture’s instruction. First strand cDNA was synthesized using Super Script IV Reverse Transcriptase (Thermo) with random primer (Thermo) and oligodT primer (Thermo). PCR reactions were performed using Quick Taq HS Dymix (TOYOBO, Japan) and pancoronaviral or specific primers (Table 1). The PCR condition was as follows: 94°C for 1 min; 40 cycles of 94°C for 30 sec, 56°C for 30 sec, and 68°C for 1 min. After PCR, amplicons were visualized by agarose gel electrophoresis staining ethidium bromide. The results are shown in Table 2. Two amplicons were detected in primer set 1 of specimen No.3 and primer set 4 of specimen No.4. The amplicons were gel-purified using Wizard SV Gel and PCR Clean-Up System (Promega) following manufacture’s instruction. The sequencing analysis was performed with Bigdye tterminator v3.1 cycle sequencing kit and Ampure XP purification. Direct sequencing analysis of the amplicons were unsuccessful maybe due to low intensity of band. Therefore, to increase the amount of template, the amplicons were re-amplified by 2nd PCR with the same enzyme and primer sets with some modification of PCR condition as follows: 94°C for 2 min; 40 cycles of 94°C for 30 sec, 52°C for 30 sec, and 68°C for 45 sec. The sequencing analysis using re-amplified template was performed and the sequence of amplicon of primer set 1 could be decoded. The 376bp of analyzed sequence showed 100% match with the sequence of WH-human1 (MN908947). HCoV229E (VR740) and MERS-CoV (EMC) could be used as positive control for pan coronavirus primer set, but there was no positive control for WN-human1 specific primers. Water only was used as negative control.