Primerscript one step rt pcr kit ver 2

The IBV strain ck/CH/LJX/2017/07 (referred as JX17) was isolated from the IBV-infected yellow feather broilers vaccinated with H120 and 4/91 strains in Jiangxi province, China, in November 2017. The infected chickens appeared typical signs of IBV infection, such as coughing, sneezing, gasping, tracheal rale and wet droppings. The viruses were propagated through blind passage five times in 9-day-old embryos of specific pathogen-free (SPF) chickens via the allantoic cavity. The allantoic fluid was examined using RT-PCR to detect the virus with PrimerScript™ One Step RT-PCR Kit Ver.2 (TaKaRa, Japan) according to the manufacturer’s instruction. PCR primers for IBV detection were described before (Zhang et al., 2018 (link)). Furthermore, we used limiting dilution passage which was described before to get monocle IBV virus (Zhang et al., 2018 (link)). Briefly, 0.2 ml allantoic fluid diluted to 10²-10⁹ times was inoculated into each embryo (4 embryos for each dilution). The allantoic fluid from the embryos inoculated with the highest dilution ratio that was positive for IBV was used for the next dilution passage. After three limiting dilution passages, the allantoic fluid positive for IBV was selected for genome sequencing and pathogenicity test. The virus isolation and follow-up experiments were carried out in biosafety level 2 laboratory.

SYBR RT-PCR was performed with the one step SYBR PrimeScript Plus RT-PCR Kit (TaKaRa Biotechnology (Dalian) Company Limited, Dalian, China) in the ABI7500 system in 20 μL reaction volume containing 2 μL total RNA, 0.8 μL forward and backward Primers (10 μM). The one step RT-PCR was carried out in a 50 μL total reaction volume by using Primerscript one step RT-PCR Kit Ver.2 (TaKaRa Biotechnology (Dalian) Company Limited, Dalian, China) with 8 μM forward and backward primers and 1 μL RNA, according to the manufacture's protocol.