The Study Of DNA Probe Detection Of The Channel Catfish Virus

The study of DNA probe detection of the channel catfish virus was conducted. The DNA probe was prepared by PCR amplification labeled with DIG, then the PCR-ELISA assay and dot blot assay for detecting CCV were established meanwhile, the results were as follows:1. A probe and a pair of primers were designed based on the ORF6 of CCV. Probe was prepared by labeling the genomic fragment(DNA)with digoxigenin. The hybridization was carried out on microplate wells pre-coated with streptoavidin and the 5' end biotin labeled-probe. Then the hybridized product was detected with a AP-labeled anti-DIG antibody and PNPP substrate.2. The optimal PCR reaction system was carried out in 25μL volumes containing 1.0μL of template DNA, 0.4 mmol/L of each primer, 4 mmol/L MgCL₂, 0.4 mmol/L dNTP, 1×PCR Buffer and 1.0 unit of Tag DNA polymerase; The mixture was pre-denatured at 94℃for 5 min, thirty amplification cycles were preformed, each cycle consisting of a denaturation step at 94℃for 1 min, annealing at 58℃for 30 sec, extension at 72℃for 45 sec and a final extension at 72℃for 10 min.3. The optimal reaction conditions of PCR-ELISA procedure: the optimal probe concentration was 50 pmol/mL; the optimal hybridization temperature was 42℃; the optimal hybridization time was 90 min.4. The results of the specificity and sensitivity detection showed that no cross-reactivity was observed when nucleic acid templates of controls were tested. The minimal detection threshold was 5 fg of the CCV DNA.5. In addition, the PCR-ELISA assay was introduced to dectect the experimental infected samples. The CCV DNA could be detected on the third to the sixth day after infection respectively. The detection results of experimental infected samples indicated that the sensitive and specific method was useful for early detection of CCV infection.6. A dot blot hybridization assay with digoxigenin labeled DNA probe was established, the detection threshold was 20 pg pT-ORF6 and the DNA probe had strong specificity. The detection technology can save the cost of testing for the diagnosis of CCV to provide more simple and efficient technology.