Development And Application Of The Rapid Detection Kit Of Classical Swine Fever Virus By Real Time Fluorogenetic Quantitative PCR Technology

In order to establish the special, sensitive and rapid CSFV diagnosis technology in our country and provide the powerful technical method for the CSF purification and the control, a rapid method is developed for detection of Classical swine fever virus (CSFV) combining real time fluorogenetic quantitative PCR technology (FQ-PCR) and the ABI detection system. Five pairs of specific CSFV fluorescence probes and primers are designed according to integrated arrangement and comparison of 29 CSFV, 18 BVDV- I and 6 BVDV-II complete gene sequences downloaded from the GenBank, at the same time skimming through homologous part together with BVDV in order to guarantee the CSFV-specific fluorescence probes. A CSFV FQ-PCR fluorogenetic quantitative RT- PCR assay for CSFV is built up through the selection from 5 pairs probes and primers using RNA templates of SM,HCLV,GDZ1/95,HeBHH1/95,BJCY1/96,JL1/94 and the optimization of the density of the reverse transcriptase, the selected upper and lower primers and probe. An uninfective RNA standard between 5'-UTR and N^pro made by transcriptase in vitro has been measured by spectrophotometer that it has a good purification with OD₂₆₀ / OD₂₈₀ value 1.9345. And there is a good line relationship between CT value and the start density of RNA template, with the correlation coefficient r 0.999, slope -3.412. It has been testified that FQ-PCR detection technique has good repetition between or in batches, with lower variation coefficient 0.66-4.42% and 0.77-4.19%. In the specificity experiment, detection rate is zero in other swine disease samples by the FQ-PCR, which validates the method has the high specificity of CSFV.As to the sensitivity, match rate is up to 88% comparing with the hog cholera immunofluorescent (HCFA) technique and more sensitive (lower detection limit) than the HCFA, also 100-times more sensitive than the conventional RT-nPCR. In addition, the reproducibility trial and stability trial for 10 monthes duration have synthesized to evaluate several indexes of the CSFV FQ- PCR detection kit that has been built up using three batches of qualified FQ-PCR reagent; three batches are 20051001, 20051002, and 20051003. CSFV FQ-PCR technique shows the superiority through comparising reaction time, CT value, fluorescence increased scope and reaction cost with one-step Ready- To- Go RT- PCR Beads and the RT-nPCR, which confirms specific, sensitive, rapid CSFV laboratory diagnostic method.A clinical diagnosis trial has been carried on 187 suspicious CSFV tissue samples from parts of provinces in our country, applying CSFV FQ-PCR, HCFA or virus separation, then analyzed results with the statistical soft manifesting that FQ- PCR positive rate of all these samples is 50.80%, HCFA is 41.27%, between these two detection techniques has significance of difference ( t=3.643, p <0.01) and with a match rate 86.24%. As for a positive result detected by CSFV FQ- PCR while HCFA show negative result, adopted sequencing the amplified product of E2 gene by RT- nPCR to prove its existing, which has established that it's possible to apply the real time FQ- PCR built up in quick, accurate diagnosis CSFV of the field samples.