African swine fever virus TaqMan fluorescent quantitative PCR detection kit and application thereof
An African swine fever virus and detection kit technology, which can be used in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., and can solve problems such as reduction of virus infectivity
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Embodiment 1
[0033] Based on the ASFV E248R gene sequence, the specific probes and primers required by Oligo7 software were used for fluorescence quantitative PCR. The upstream primer sequence was GGAGGCTCTACAAGCAAA, the downstream primer sequence was CATCACCGAATACGCCTA, and the probe sequence was FAM-AATACGACCAACATTATCAGCAAT-BHQ1. The above probe and primer sequences have been analyzed by BLAST as ASFV-specific sequences, and the regions where the probe primers are located are relatively conservative compared with other strains (see Figure 2-1 ). The probes were synthesized by Shanghai Gema Pharmaceutical Technology Co., Ltd., and the primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd.
[0034] Fluorescence quantitative PCR detection was carried out using Probe qPCR Mix (2×) enzyme 20 μl system (see Table 1), and the reaction cycle conditions were: pre-denaturation at 95 °C for 1 min; denaturation at 95 °C for 5s, annealing for 30s, 40 cycles, each cycle After the end, th...
Embodiment 2
[0038] According to the optimized fluorescent quantitative PCR reaction conditions, the prepared standard is subjected to the fluorescent quantitative PCR test, and each gradient is set to three replicates, which are automatically generated by the fluorescent quantitative PCR instrument.
[0039] with 10 5 , 10 6 , 10 7 Copies / μL of standard samples were subjected to real-time quantitative PCR experiments at the same time, and three replicates were set for each gradient, and the Ct values were calculated to calculate the within-group standard deviation and coefficient of variation (CV). with 10 5 , 10 6 , 10 7The standard of copies / μL was subjected to three quantitative PCR experiments at different times, and the Ct values obtained were used to calculate the standard deviation and coefficient of variation between groups. The reproducibility of the real-time PCR method was assessed using the coefficients of variation between and within groups (Table 2, Table 3).
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Embodiment 3
[0046] with 1×10 1 -1×10 8 Sensitivity test was carried out with the standard substance of copies / μL, 3 replicates were set for each gradient, the minimum detection concentration of the method was determined by observing the standard curve, and the Ct value was the ordinate and the number of copies was the ordinate, and a standard curve was established to evaluate its linear relationship, verifying the sensitivity of the method (see figure 1 ) The minimum detection amount of this method is 10 copies, and the amplification curve can appear when the sample concentration is greater than 10 copies / μL, and the standard curve is automatically generated by the Roche Fluorometer (see figure 2 ). The standard curve equation is y = -3.431x + 41.31 and R² = 1.00. The experimental results show that the ASFV fluorescence quantitative detection method established in this experiment has high sensitivity.
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