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Virologica Sinica, 18 (2) : 124-128, 2003
Development of a Fluorogenic Quantitative PCR Assay for Rapid Quantification ofHog cholera lapmizea virus
College ofLife Science,Wuhan University,Wuhan 430072,China
2.Wuhan Chopper Biochemistry Limited Company, Wuhan 430072,China
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
  Published online: 15 Apr 2003
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