Title: Development and Application of A Fluorescent Quantitative PCR Assay for the Detection of Haempohlius Parasuis
Abstract: A fluorescent quantitative PCR based on SYBR Green I was developed for the detection of Haempohlius parasuis.A pair of primers were designed specificly for infB gene of HPS according to the published nucleotide sequence(GenBank accession No.:DQ781808.1).The expected fragment was amplified using PCR from extracted DNA of HPS culture by boiling.The PCR product of target gene was cloned into pMD18-T vector and then transferred into DH5α.Positive recombinant plasmid was selected and used as a positive quantitative template to establish a standard curve.The results showed that the fluorescence quantitative PCR assay was able to detect HPS and had no cross-reaction with other bacteria from swine.The sensibility of this assay attained 10 copies of plasmid DNA,which was 100 times higher than routine PCR.The C.V.of the intra or inter was no more than 2.5%,indicating that the developed fluorescence quantitive PCR is a stable assay.Clinical detection showed development of the method achieved the rapid diagnosis and quantitative detection of HPS.
Publication Year: 2010
Publication Date: 2010-01-01
Language: en
Type: article
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