title: A direct real-time polymerase chain reaction assay for rapid high-throughput detection of highly pathogenic North American porcine reproductive and respiratory syndrome virus in China without RNA purification.
abstract: BACKGROUND
  Porcine reproductive and respiratory syndrome virus (PRRSV), and particularly its highly pathogenic genotype (HP-PRRSV), have caused massive economic losses to the global swine industry.
  
  
  RESULTS
  To rapidly identify HP-PRRSV, we developed a direct real-time reverse transcription polymerase chain reaction method (dRT-PCR) that could detect the virus from serum specimen without the need of RNA purification. Our dRT-PCR assay can be completed in 1.5 h from when a sample is received to obtaining a result. Additionally, the sensitivity of dRT-PCR matched that of conventional reverse transcription PCR (cRT-PCR) that used purified RNA. The lowest detection limit of HP-PRRSV was 6.3 TCID50 using dRT-PCR. We applied dRT-PCR assay to 144 field samples and the results showed strong consistency with those obtained by cRT-PCR. Moreover, the dRT-PCR method was able to tolerate 5-20% (v/v) serum.
  
  
  CONCLUSIONS
  Our dRT-PCR assay allows for easier, faster, more cost-effective and higher throughput detection of HP-PRRSV compared with cRT-PCR methods. To the best of our knowledge, this is the first report to describe a real-time RT-PCR assay capable of detecting PRRSV in crude serum samples without the requirement for purifying RNA. We believe our approach has a great potential for application to other RNA viruses.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100 China ; College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, 518060 China.
authors.forename: Kang
authors.initials: K
authors.lastname: Kang
authors.affiliation: Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064 China.
authors.forename: Keli
authors.initials: K
authors.lastname: Yang
authors.affiliation: College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, 518060 China.
authors.forename: Jiasheng
authors.initials: J
authors.lastname: Zhong
authors.affiliation: Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064 China.
authors.forename: Yongxiang
authors.initials: Y
authors.lastname: Tian
authors.affiliation: College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, 518060 China.
authors.forename: Limin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Shenzhen Ao Dong Inspection and Testing Technology Co,. Ltd, Shenzhen, 518000 China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Zhai
authors.affiliation: Shenzhen Ao Dong Inspection and Testing Technology Co,. Ltd, Shenzhen, 518000 China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Veterinary Medicine Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640 China.
authors.forename: Changxu
authors.initials: C
authors.lastname: Song
authors.affiliation: Northwestern University, Evanston, IL USA.
authors.forename: Christine Yuan
authors.initials: CY
authors.lastname: Gou
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100 China.
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.affiliation: College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen, 518060 China.
authors.forename: Deming
authors.initials: D
authors.lastname: Gou
keywords: Highly pathogenic; Porcine reproductive and respiratory syndrome virus; Real-time RT-PCR