Five kinds of porcine diarrhea virus enrichment multiplex PCR rapid detection kit and its application
A kit and porcine diarrhea technology, applied in the field of multiplex PCR rapid diagnostic kits, can solve the problems of misjudgment and misjudgment, inconsistent amplification efficiency, and inability to meet the early and rapid detection of viruses, achieve high specificity, and improve clinical virus detection Horizontal, wide-ranging effects
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Embodiment 1
[0063] Embodiment 1, kit structure
[0064] An enrichment multiplex PCR rapid diagnostic kit for simultaneous and specific detection of five porcine diarrhea virus infections, including: PCR reaction solution, primers, GCR virus standard, GAR virus standard, TGEV virus standard, PEDV virus standard, PCV2 virus Composition of standard, positive control, negative control and box.
[0065] There are container holes in the box body, respectively place PCR reaction solution tubes, primer tubes, GCR virus standard tubes, GAR virus standard tubes, TGEV virus standard tubes, PEDV virus standard tubes, PCV2 virus standard tubes, positive Control quality control and negative control quality control.
[0066] Wherein the PCR reaction solution comprises PCR reaction buffer (containing magnesium chloride, deoxyribonucleotide triphosphate mixture, etc.) and Taq DNA polymerase, and the primer tubes are five kinds of porcine diarrhea virus-specific combination primers, five kinds of virus-sp...
Embodiment 2
[0067] Embodiment 2, preparation of plasmid standard
[0068] Step 1: Primer Synthesis
[0069] The 5 pairs of virus-specific primers designed in the present invention (see Table 1) were synthesized by Shanghai Sangon Biology Technology Service Company, and the synthesis amount was 3 OD per primer.
[0070] Step 2: Total viral DNA / RNA extraction
[0071] Place the samples containing TGEV, GAR, GCR, PEDV and PCV2 virus sources in a 1.5mL centrifuge tube, and use the AxyPrep Body Fluid Virus DNA / RNA Mini Kit (AXYGEN) to extract viral nucleic acid from the samples according to the product instruction manual to obtain Viral DNA / RNA.
[0072] Step 3: Reverse transcription reaction
[0073] Add 1 μl of the viral DNA / RNA template prepared in the previous step to an RNase-free 0.2ml PCR tube, and use TaKaRa RNA PCR Kit (AMV) Ver.3.0 (Code No.RR019A) for RT-PCR reaction, in which 1 μl, 25 mM MgCl 2 2μl, 10mM dNTP Mixture 1μl, 40U / μl RNase Inhibitor0.25μl, 5U / μl AMV RTase XL0.5μl,...
Embodiment 3
[0078] Embodiment 3 The present invention detects five kinds of porcine diarrhea virus specific experiments
[0079] Step 1: Primer Synthesis
[0080] The 5 pairs of virus-specific combined primers and 1 pair of universal primers (see Table 1) designed by the present invention were synthesized by Shanghai Sangon Biology Technology Service Company, and the synthesis amount was 3 OD per primer. .
[0081] The second step: specific detection
[0082] Prepare 20 copies of the same reaction solution according to the enrichment multiplex PCR reaction system, that is, 10×PCR buffer 2.0μL, 25mMMgCl 2 2.0 μL, 2.5 mM dNTP 1.6 μL, Taq DNA Polymerase 2.5U, 5 pairs of virus-specific combined primers and 1 pair of universal primers in Table 1, where the final concentration of GCR, GAR, TGEV, PEDV and PCV2 upstream and downstream combined primers is 0.01 μM , the upstream and downstream primers of the universal primer were 0.5μM, and 1.0×10 4 Copies of GCR, GAR, TGEV, PEDV, PCV2, GCR+GA...
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