Identification of Adenovirus, Influenza Virus, Parainfluenza Virus, and Respiratory Syncytial Virus by Two Kinds of Multiplex Polymerase Chain Reaction (PCR) and a Shell Vial Culture in Pediatric Patients with Viral Pneumonia

Lee, Jong-Han; Chun, Jin-Kyong; Kim, Dong Soo; Park, Yongjung; Choi, Jong Rak; Kim, Hyon-Suk

Seeplex TM RV detection kit (http://www.seegene.co.kr/en/index.php) Nucleic acids extracted from nasopharyngeal aspirates were used for the synthesis of first-strand cDNAs by Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The Seeplex TM RV detection kit (Cosmo Genetech, Seoul, Korea) contained A and B sets of primers designed by highly conserved regions of genetic sequences for the 12 respiratory viruses. The Seeplex TM kit is designed to identify AdV, human metapneumovirus (hMPV), Human coronavirus (HCoV) 229E/NL63, parainfluenza virus (PIFV) 1, PIFV 2, and PIFV 3, and the Seeplex TM RV detection kit B is designed to detect IFV A, IFV B, RSV A, RSV B, rhinovirus (RhV) , and HCoV OC43/ HKU1. Each PCR was conducted in a final reaction volume of 20 µL containing 3 µL of cDNA, 3 µL of 8-methoxypsoralen (MOP) solution, 4 µL of 5×RV Primer, and 10 µL of 2×master mix. The PCR protocol was 94˚C for 30 sec, followed by 35 cycles of 60˚C for 1.5 min, and 72˚C for 1.5 min, followed by a 10 min final extension at 72˚C. The amplified products were separated on a 2% agarose gel stained with ethidium bromide. Each run included a molecular size marker and internal control. Also, American Type Culture Collection (ATCC) standard viruses were used for positive control and 10 µL distilled water as negative control.