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.
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 TCID(50) 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.
Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 427 Maduan St., Nangang District, 150001, Harbin, China.
Zheng
Z
Chai
Wenjun
W
Ma
Fang
F
Fu
Yuekun
Y
Lang
Wei
W
Wang
Guangzhi
G
Tong
Qinfang
Q
Liu
Xuehui
X
Cai
Xi
X
Li
D000818:Animals; D052160:Benzothiazoles; D002681:China; D003959:Diamines; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000191:economics / Q000379:methods; D013552:Swine; D013997:Time Factors; D044366:Transition Temperature; D014730:Veterinary Medicine / Q000191:economics / Q000379:methods*; D014773:Virology / Q000191:economics / Q000379:methods*
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I