Development of a Fluorogenic Quantitative PCR Assay for Rapid Quantification ofHog cholera lapmizea virus

A rapid an d reproducible method was first established for assessment of Hog cholera lapinized virus(HCLV)loads in Classical Swine Fever vaccine using Flurogenic quantitative PCR (FQ·PCR)combines LiIghtCycler sequence detection system．The method contains a pair of primers and an internal daul·labled fluorogenic probe spanning the part of 5’noncoding region(5’NCR)of HCLV, the use of such a probe combined with the 5’-3’nuclease activity of Taq polymerase allows direct quan tification of the PCR product by the detection of a fluorescent reporter released in th e course of the exponential phase of the PCR．Th e sensitivity of the assay was 10 copies per reaction．Th e assay is linear within 6-log dynamic rang．The coeficient of variation(CV)of the standard of Ct value is 2．3％ 一5．1％ (n=10)；The CV ofvaccine sample is 0．85％一2．8％ in intra—assay and 2．5％一7．3％ in inter·assay(n =5)，respectively；Th e CV of the same sam ple in diferent RNA isolation and reverse transcription iS 5．0 ％ (n=5)．Nine vaccines were quantified by this method and give similar but more accurate results compared to the conventional rabbit fever reaction．Th e entire assay,including RNA isolation，reverse tran scription，an d quan tification，could be completed within 4 hour s．In conclusion，the high sensitivity， simplicity,an d reproducibility of the HCLV RNA quan tification which allows the screening of large numbers of sam ples，combined with its wide dynamic ran g，makes this method especially suitable for evaluating th e viral loads an d guiding how to confect th e vaccine，it also provides a novel an d simple research tool for CSFV