RT-PCR primer group and RT-PCR kit for simultaneously detecting four porcine RNA viruses
A primer set, swine fever virus technology, applied in DNA/RNA fragments, recombinant DNA technology, microorganisms, etc., can solve the problems of complicated operation methods, uncertain results, long diagnosis time, etc., and achieve simple detection procedures, low cost, highly specific effect
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Embodiment 1
[0039] The establishment of embodiment 1 multiplex RT-PCR method
[0040] 1. Experimental method
[0041] 1.1PCR primer design
[0042] According to some known sequences of PRRSV (Genbank No. NC 001961), TGEV (Genbank No. NC 002306), PEDV (Genbank No. NC 003436), and CSFV (GenBank No. EU497410) published by GenBank, several pairs of primers were designed, as shown in Table 1 and Table 2. Use DNAstar to analyze the primer-dimers of the 4 virus primers designed to avoid the formation of stable primer-dimers between the primers, and analyze the homology or complementarity of the amplified sequences of each pair of primers to avoid them. There is a high homology or complementarity between them, and the two sets of primers in Table 1 and Table 2 are determined by specificity tests to determine the multiplex PCR primers for the 4 viruses.
[0043] Table 1 Primer design for multiplex RT-PCR
[0044]
[0045] Table 2 Primer design for multiplex RT-PCR
[0046]
[0047] 1.2 ...
experiment example 1
[0064] Specificity experiment of experimental example 1 primer set
[0065] 1. Experimental method
[0066] 1.1 Extraction of PPV and PRV DNA (CTAB-NaCl method)
[0067] 1) Take 0.5mL sample and place it in a sterile microcentrifuge tube, add 100μL NET buffer (1mmol / L EDTA, 10mmol / L Tris-Cl) and 50μL 2.6mol / L NaOH as denaturant and mix well.
[0068] 2) Place in a water bath at 80°C for 10 minutes. Cool to room temperature, add 20 μL of proteinase K (20 mg / mL) to a final concentration of 650 μg / mL, and act in a 37 °C water bath for 1 h.
[0069] 3) Add 100 μL of 5 mol / L NaCl and 80 μL of CTAB-NaCl solution, mix well, and act in a water bath at 65° C. for 10 minutes.
[0070] 4) Add an equal volume of chloroform-isoamyl alcohol (24:1), mix well, centrifuge at 12000r / min for 4-5min, and transfer the supernatant to a new centrifuge tube.
[0071] 5) Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix well, centrifuge at 12000r / min for 4-5min, and transfer ...
experiment example 2
[0093] Experimental Example 2 Sensitivity Experiment of Multiplex RT-PCR
[0094] 1. Experimental method
[0095] The cDNA concentrations of PRRSV, TGEV, PEDV, and CSFV obtained in Example 1 were measured by spectrophotometer OD to be 10000 μg / μL, 15000 μg / μL, 12000 μg / μL and 10000 μg / μL, respectively. The concentration was adjusted to 3 μg / μL before the experiment.
[0096] The cDNA of PRRSV, TGEV, PEDV and CSFV was diluted 10 times (from 3×10 2 ng to 3×10 -3 ng), the volume ratio of the four viral cDNAs is 1:1:1:1, amplified according to the multiplex RT-PCR amplification system and amplification program established in Example 1, and the amplification results were detected by electrophoresis.
[0097] 2. Experimental results
[0098] Amplification results see Figure 4 . It can be seen that when the mixed cDNA content of viruses PRRSV, TGEV, PEDV and CSFV is greater than 3ng, the four viruses (PRRSV, TGEV, PEDV and CSFV) can be effectively amplified.
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