PMID- 18054369
OWN - NLM
STAT- MEDLINE
DCOM- 20081028
LR  - 20220408
IS  - 0034-5288 (Print)
IS  - 0034-5288 (Linking)
VI  - 85
IP  - 1
DP  - 2008 Aug
TI  - 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.
PG  - 184-93
AB  - 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.
FAU - Martinez, E
AU  - Martinez E
AD  - Laboratorios HIPRA, S.A., Av. La Selva 135, 17170 Amer Girona, Spain.
FAU - Riera, P
AU  - Riera P
FAU - Sitja, M
AU  - Sitja M
FAU - Fang, Y
AU  - Fang Y
FAU - Oliveira, S
AU  - Oliveira S
FAU - Maldonado, J
AU  - Maldonado J
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20071203
PL  - England
TA  - Res Vet Sci
JT  - Research in veterinary science
JID - 0401300
RN  - 0 (Benzothiazoles)
RN  - 0 (Diamines)
RN  - 0 (Organic Chemicals)
RN  - 0 (Quinolines)
RN  - 163795-75-3 (SYBR Green I)
SB  - IM
MH  - Animals
MH  - Benzothiazoles
MH  - Diamines
MH  - Genotype
MH  - *Organic Chemicals
MH  - Phylogeny
MH  - Polymerase Chain Reaction
MH  - Porcine Reproductive and Respiratory Syndrome/diagnosis/*virology
MH  - Porcine respiratory and reproductive syndrome virus/*genetics/*isolation & 
      purification
MH  - Quinolines
MH  - Reproducibility of Results
MH  - Reverse Transcriptase Polymerase Chain Reaction/*veterinary
MH  - Sensitivity and Specificity
MH  - Swine
EDAT- 2007/12/07 09:00
MHDA- 2008/10/29 09:00
CRDT- 2007/12/07 09:00
PHST- 2006/04/11 00:00 [received]
PHST- 2007/04/23 00:00 [revised]
PHST- 2007/10/11 00:00 [accepted]
PHST- 2007/12/07 09:00 [pubmed]
PHST- 2008/10/29 09:00 [medline]
PHST- 2007/12/07 09:00 [entrez]
AID - S0034-5288(07)00237-8 [pii]
AID - 10.1016/j.rvsc.2007.10.003 [doi]
PST - ppublish
SO  - Res Vet Sci. 2008 Aug;85(1):184-93. doi: 10.1016/j.rvsc.2007.10.003. Epub 2007 
      Dec 3.