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

Journal of Animal Science and Biotechnology

Deming Gou³  Jun Luo¹  Christine Yuan Gou⁴  Changxu Song⁶  Li Zhang⁵  Jianxin Zhai⁵  Limin Zhang³  Yongxiang Tian²  Jiasheng Zhong³  Keli Yang²  Kang Kang³ 

[1] College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China;Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China;College of Life Sciences, Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen University, Shenzhen 518060, China;Northwestern University, Evanston, IL, USA;Shenzhen Ao Dong Inspection and Testing Technology Co,. Ltd, Shenzhen 518000, China;Veterinary Medicine Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China

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

Others  :  1135891
DOI  :  10.1186/2049-1891-5-45

 received in 2014-05-12, accepted in 2014-09-25,  发布年份 2014

【 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.

【 授权许可】

   
2014 Kang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Rossow KD: Porcine reproductive and respiratory syndrome. Vet Pathol 1998, 35:1-20.
• [2]Albina E: Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet Microbiol 1997, 55:309-316.
• [3]Cavanagh D: Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 1997, 142:629-633.
• [4]Elazhary Y, Weber J, Bikour H, Morin M, Girard C: ‘Mystery swine disease’ in Canada. Vet Rec 1991, 129:495-496.
• [5]Wensvoort G, Terpstra C, Pol JM, ter Laak EA, Bloemraad M, de Kluyver EP, Kragten C, van Buiten L, den Besten A, Wagenaar F: Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet Q 1991, 13:121-130.
• [6]Tian K, Yu X, Zhao T, Feng Y, Cao Z, Wang C, Hu Y, Chen X, Hu D, Tian X, Liu D, Zhang S, Deng X, Ding Y, Yang L, Zhang Y, Xiao H, Qiao M, Wang B, Hou L, Wang X, Yang X, Kang L, Sun M, Jin P, Wang S, Kitamura Y, Yan J, Gao GF: Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS One 2007, 2:e526.
• [7]Mengeling WL, Lager KM, Wesley RD, Clouser DF, Vorwald AC, Roof MB: Diagnostic implications of concurrent inoculation with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus. Am J Vet Res 1999, 60:119-122.
• [8]Nodelijk G, Wensvoort G, Kroese B, van Leengoed L, Colijn E, Verheijden J: Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus. Vet Microbiol 1996, 49:285-295.
• [9]Albina E, Leforban Y, Baron T, Plana Duran JP, Vannier P: An enzyme linked immunosorbent assay (ELISA) for the detection of antibodies to the porcine reproductive and respiratory syndrome (PRRS) virus. Ann Rech Vet 1992, 23:167-176.
• [10]Dea S, Wilson L, Therrien D, Cornaglia E: Detection of antibodies to the nucleocapsid protein of PRRS virus by a competitive ELISA. Adv Exp Med Biol 2001, 494:401-405.
• [11]Chai Z, Ma W, Fu F, Lang Y, Wang W, Tong G, Liu Q, Cai X, Li X: A SYBR Green-based real-time RT-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China. Arch Virol 2013, 158:407-415.
• [12]Wernike K, Hoffmann B, Dauber M, Lange E, Schirrmeier H, Beer M: Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time rt-PCR. PLoS One 2012, 7:e38251.
• [13]Chen NH, Chen XZ, Hu DM, Yu XL, Wang LL, Han W, Wu JJ, Cao Z, Wang CB, Zhang Q, Wang BY, Tian KG: Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR. J Virol Methods 2009, 161:192-198.
• [14]Xiao XL, Wu H, Yu YG, Cheng BZ, Yang XQ, Chen G, Liu DM, Li XF: Rapid detection of a highly virulent Chinese-type isolate of Porcine Reproductive and Respiratory Syndrome Virus by real-time reverse transcriptase PCR. J Virol Methods 2008, 149:49-55.
• [15]Yang K, Li Y, Duan Z, Guo R, Liu Z, Zhou D, Yuan F, Tian Y: A one-step RT-PCR assay to detect and discriminate porcine reproductive and respiratory syndrome viruses in clinical specimens. Gene 2013, 531:199-204.
• [16]Queipo-Ortuno MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P: Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clin Vaccine Immunol 2008, 15:293-296.
• [17]Al-Soud WA, Radstrom P: Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 2001, 39:485-493.
• [18]Tsai YL, Olson BH: Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol 1992, 58:2292-2295.
• [19]Watson RJ, Blackwell B: Purification and characterization of a common soil component which inhibits the polymerase chain reaction. Can J Microbiol 2000, 46:633-642.
• [20]Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM: Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res 2009, 37:e40.
• [21]Henke W, Herdel K, Jung K, Schnorr D, Loening SA: Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res 1997, 25:3957-3958.
• [22]Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I: Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn 2006, 8:544-550.
• [23]Zhang Z, Kermekchiev MB, Barnes WM: Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 2010, 12:152-161.
• [24]Petry H, Bachmann B, Luke W, Hunsmann G: PCR sequencing with the aid of detergents. Methods Mol Biol 1996, 65:105-109.
• [25]Nishimura N, Nakayama H, Yoshizumi S, Miyoshi M, Tonoike H, Shirasaki Y, Kojima K, Ishida S: Detection of noroviruses in fecal specimens by direct RT-PCR without RNA purification. J Virol Methods 2010, 163:282-286.
• [26]Bachofen C, Willoughby K, Zadoks R, Burr P, Mellor D, Russell GC: Direct RT-PCR from serum enables fast and cost-effective phylogenetic analysis of bovine viral diarrhoea virus. J Virol Methods 2013, 190:1-3.
• [27]Pastorino B, Bessaud M, Grandadam M, Murri S, Tolou HJ, Peyrefitte CN: Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses. J Virol Methods 2005, 124:65-71.