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.
The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03TCID(50)/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature (T(m)) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T(m) values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.
Laboratorios HIPRA, S.A., Av. La Selva 135, 17170 Amer Girona, Spain.
E
E
Martínez
P
P
Riera
M
M
Sitjà
Y
Y
Fang
S
S
Oliveira
J
J
Maldonado
D000818:Animals; D052160:Benzothiazoles; D003959:Diamines; D005838:Genotype; D009930:Organic Chemicals; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D019318:Porcine Reproductive and Respiratory Syndrome / Q000175:diagnosis / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000302:isolation & purification*; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013552:Swine
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I