Abstract
SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID50 for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.
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References
Rossow KD (1998) Porcine reproductive and respiratory syndrome. Vet Pathol 35:1–20
Cavanagh D (1997) Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 142:629–633
Murtaugh MP, Elam MR, Kakach LT (1995) Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus. Arch Virol 140:1451–1460
Nelson EA, Christopher-Hennings J, Drew T, Wensvoort G, Collins JE, Benfield DA (1993) Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol 31:3184–3189
Meulenberg JJ, Hulst MM, de Meijer EJ, Moonen PL, den Besten A, de Kluyver EP, Wensvoort G, Moormann RJ (1993) Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192:62–72
Zhou L, Zhang J, Zeng J, Yin S, Li Y, Zheng L, Guo X, Ge X, Yang H (2009) The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence. J Virol 83:5156–5167
Zhou L, Chen S, Zhang J, Zeng J, Guo X, Ge X, Zhang D, Yang H (2009) Molecular variation analysis of porcine reproductive and respiratory syndrome virus in China. Virus Res 145:97–105
Zhou Z, Ni J, Cao Z, Han X, Xia Y, Zi Z, Ning K, Liu Q, Cai L, Qiu P, Deng X, Hu D, Zhang Q, Fan Y, Wu J, Wang L, Zhang M, Yu X, Zhai X, Tian K (2011) The epidemic status and genetic diversity of 14 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolates from China in 2009. Vet Microbiol 150:257–269
Ni J, Yang S, Bounlom D, Yu X, Zhou Z, Song J, Khamphouth V, Vatthana T, Tian K (2012) Emergence and pathogenicity of highly pathogenic Porcine reproductive and respiratory syndrome virus in Vientiane, Lao People’s Democratic Republic. J Vet Diagn Invest 24:349–354
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 (2007) Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE 2:e526
Tong GZ, Zhou YJ, Hao XF, Tian ZJ, An TQ, Qiu HJ (2007) Highly pathogenic porcine reproductive and respiratory syndrome, China. Emerg Infect Dis 13:1434–1436
Li Y, Wang X, Bo K, Wang X, Tang B, Yang B, Jiang W, Jiang P (2007) Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China. Vet J 174:577–584
Li B, Fang L, Liu S, Zhao F, Jiang Y, He K, Chen H, Xiao S (2010) The genomic diversity of Chinese porcine reproductive and respiratory syndrome virus isolates from 1996 to 2009. Vet Microbiol 146:226–237
Li B, Fang L, Guo X, Gao J, Song T, Bi J, He K, Chen H, Xiao S (2011) Epidemiology and evolutionary characteristics of the porcine reproductive and respiratory syndrome virus in China between 2006 and 2010. J Clin Microbiol 49:3175–3183
Yu X, Chen N, Wang L, Wu J, Zhou Z, Ni J, Li X, Zhai X, Shi J, Tian K (2012) New genomic characteristics of highly pathogenic porcine reproductive and respiratory syndrome viruses do not lead to significant changes in pathogenicity. Vet Microbiol 158:291–299
Van Woensel P, Van Der Wouw J, Visser N (1994) Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction. J Virol Methods 47:273–278
Martinez E, Riera P, Sitja M, Fang Y, Oliveira S, Maldonado J (2008) Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res Vet Sci 85:184–193
Balka G, Hornyak A, Balint A, Benyeda Z, Rusvai M (2009) Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus. J Virol Methods 158:41–45
Li Q, Q-f Zhou, C-y Xue, J-y Ma, D-z Zhu, Y-c Cao (2009) Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification assay. J Virol Methods 155:55–60
Xiao XL, Wu H, Yu YG, Cheng BZ, Yang XQ, Chen G, Liu DM, Li XF (2008) 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 149:49–55
Zhou YJ, Hao XF, Tian ZJ, Tong GZ, Yoo D, An TQ, Zhou T, Li GX, Qiu HJ, Wei TC, Yuan XF (2008) Highly virulent porcine reproductive and respiratory syndrome virus emerged in China. Transbound Emerg Dis 55:152–164
Li XS, Fu F, Lang YK, Li HZ, Wang W, Chen X, Tian HB, Zhou YJ, Tong GZ, Li X (2011) Development and preliminary application of an immunochromatographic strip for rapid detection of infection with porcine reproductive and respiratory syndrome virus in swine. J Virol Methods 176:46–52
Tam S, Clavijo A, Engelhard EK, Thurmond MC (2009) Fluorescence-based multiplex real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth disease virus. J Virol Methods 161:183–191
Varga A, James D (2005) Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing. J Virol Methods 123:213–220
Papp AC, Pinsonneault JK, Cooke G, Sadee W (2003) Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. Biotechniques 34:1068–1072
Pérez LJ, Perera CL, Frías MT, Núñez JI, Ganges L, de Arce HD (2012) A multiple SYBR Green I-based real-time PCR system for the simultaneous detection of porcine circovirus type 2, porcine parvovirus, pseudorabies virus and Torque teno sus virus 1 and 2 in pigs. J Virol Methods 179:233–241
Belak S (2007) Molecular diagnosis of viral diseases, present trends and future aspects: a view from the OIE collaborating centre for the application of polymerase chain reaction methods for diagnosis of viral diseases in veterinary medicine. Vaccine 25:5444–5452
Gagnon CA, del Castillo JR, Music N, Fontaine G, Harel J, Tremblay D (2008) Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey. J Vet Diagn Invest 20:545–558
Templeton KE, Scheltinga SA, Beersma MFC, Kroes ACM, Claas ECJ (2004) Rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 42:1564–1569
Lehmann LE, Hunfeld K-P, Emrich T, Haberhausen G, Wissing H, Hoeft A, Stüber F (2008) A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Med Microbiol Immunol 197:313–324
Brittain-Long R, Nord S, Olofsson S, Westin J, Anderson L-M, Lindh M (2008) Multiplex real-time PCR for detection of respiratory tract infections. J Clin Virol 41:53–56
Huang YW, Dryman BA, Harrall KK, Vaughn EM, Roof MB, Meng XJ (2010) Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses. J Virol Methods 170:140–146
Egli C, Thür B, Liu L, Hofmann MA (2001) Quantitative TaqMan® RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus. J Virol Methods 98:63–75
Chen NH, Chen XZ, Hu DM, Yu XL, Wang LL, Han W, Wu JJ, Cao Z, Wang CB, Zhang Q, Wang BY, Tian KG (2009) 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 161:192–198
Chen N, Cao Z, Yu X, Deng X, Zhao T, Wang L, Liu Q, Li X, Tian K (2011) Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China. J Gen Virol 92:880–892
Wernike K, Bonilauri P, Dauber M, Errington J, Leblanc N, Revilla-Fernández S, Hjulsager C, Isaksson M, Stadejek T, Beer M, Hoffmann B (2012) Porcine reproductive and respiratory syndrome virus: interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods. J Vet Diagn Invest 24:855–866
Wernike K, Hoffmann B, Dauber M, Lange E, Schirrmeier H, Beer M (2012) Detection and typing of highly pathogenic porcine reproductive and respiratory syndrome virus by multiplex real-time RT-PCR. PLoS ONE 7:e38251
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This study was supported by grants from Major International Scientific Cooperation Projects in the National Animal Zoonoses Prevention and Control of Key Technologies (2010DFB33920).
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Chai, Z., Ma, W., Fu, F. et al. 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 158, 407–415 (2013). https://doi.org/10.1007/s00705-012-1504-7
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DOI: https://doi.org/10.1007/s00705-012-1504-7