A one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe.
The aim of this study was to develop a one-step real-time reverse transcription-polymerase chain reaction assay using the minor groove binding probe (MGB rRT-PCR) for rapid and quantitative detection of classical swine fever virus (CSFV). The method, which targets the 5'-nontranslated region (5'NTR) of the viral genome, detected all CSFV isolate tested, but not heterologous pathogens. Using an in vitro transcript of the 5'NTR as a quantitative standard for the CSFV genome copy number, the assay had a detection limit of 10 copies/reaction, and the standard curve had a linear range from 10 to 10(7) copies/reaction, with good reproducibility. As determined by an end-point dilution comparison, in most case, the sensitivity of the MGB rRT-PCR was approximately 10-fold higher than that of virus isolation and the rRT-PCR using the standard Taqman probe (standard rRT-PCR). The agreement between the MGB rRT-PCR and standard rRT-PCR, or virus isolation was 93.3% and 76.7%, respectively, when detecting 261 field samples. Due to its rapidity, high specificity and sensitivity, the MGB rRT-PCR assay provides a valuable tool for diagnosis and molecular studies of CSFV biology.
Wen
Guoyuan
G
Institute of Animal Husbandry and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan, China.
Yang
Jun
J
Luo
Qingping
Q
Hu
Zhibin
Z
Song
Nianhua
N
Zhang
Rongrong
R
Wang
Hongling
H
Ai
Diyun
D
Luo
Ling
L
Shao
Huabin
H
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D006692:Classical Swine Fever Virus / Q000235:genetics* / Q000302:isolation & purification; D017124:Conserved Sequence; D017931:DNA Primers; D016679:Genome, Viral; D007668:Kidney / Q000821:virology; D018482:Muscle, Skeletal / Q000821:virology; D009297:Nasal Mucosa / Q000821:virology; D014066:Palatine Tonsil / Q000821:virology; D018182:Pestivirus Infections / Q000175:diagnosis / Q000662:veterinary*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013045:Species Specificity; D013154:Spleen / Q000821:virology; D013552:Swine / Q000145:classification / Q000235:genetics; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral
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