| Abstrakt: |
According to the published chronic bee paralysis virus(CBPV)gene sequences, three specific primers were designed. Establish CBPV semi nested PCR detection method, the outer primer annealing temperatures(52,54,56 and 58℃),the Inner primer annealing temperatures(48,50,52 and 54℃),primer concentrations(0.1,0.2and 0.4 mmol/L)and volume of ExTaq enzyme (0.25,0.5and 1μL) for semi nested PCR were optimized, and the optimized method was verified for specificity and sensitivity. At the same time, Twenty clinical samples were tested by the developed semi nested PCR. The results show that the semi nested PCR outer primer annealing temperature, inner primer annealing temperature, primer concentration and volume of ExTaq enzyme were 56℃,50℃,0.2mmol/L and 0.25μL;no cross reactions with the cDNAs of healthy, CBPV, ABPV, CSBV, BQCV, DWV were observed by the developed semi nested PCR, with a minimun detection limit of 10-3 pg;4samples were positive from the 20 clinical samples. The established semi nested PCR detection was proved to be rapid, sensitive, specific, etc, which enable it a promising clinical diagnostic and epidemiological investigation method. |
