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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

文献类型： 外文期刊

作者： Kang, Kang 1 ; Yang, Keli 3 ; Zhong, Jiasheng 2 ; Tian, Yongxiang 3 ; Zhang, Limin 2 ; Zhai, Jianxin 4 ; Zhang, Li 4 ; So 1 ;

作者机构： 1.Northwest A&F Univ, Coll Anim Sci & Technol, Yangling 712100, Shaanxi, Peoples R China

2.Shenzhen Univ, Coll Life Sci, Shenzhen Key Lab Microbial Genet Engn, Shenzhen 518060, Peoples R China

3.Hubei Acad Agr Sci, Inst Anim Husb & Vet, Hubei Key Lab Anim Embryo & Mol Breeding, Wuhan 430064, Peoples R China

4.Shenzhen Ao Dong Inspect & Testing Technol Co Ltd, Shenzhen 518000, Peoples R China

5.Guangdong Acad Agr Sci, Vet Med Inst

关键词： Highly pathogenic;Porcine reproductive and respiratory syndrome virus;Real-time RT-PCR

期刊名称：JOURNAL OF ANIMAL SCIENCE AND BIOTECHNOLOGY （ 影响因子：5.032； 五年影响因子：5.922 ）

ISSN： 2049-1891

年卷期： 2014 年 5 卷

页码：

收录情况： SCI

摘要： 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.

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