Liquid chip kit for six porcine reproductive disorder viruses
The technology of a kit and virus liquid is applied in the field of six kinds of porcine reproductive disorder virus liquid phase chip kits, which can solve the problems of heavy workload, high cost, and long time consumption, and achieve a short detection cycle, short detection time, and high sensitivity. Effect
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Embodiment 1
[0031] Embodiment 1 kit design principle
[0032] The present invention first collects the PPV structural protein VP2 gene, the ORF2 open reading frame sequence of PCV, the gB gene of PRV, the NSP2 gene of PRRSV, the E2 gene sequence of CSFV and the M gene of JEV in GenBank according to the difference of genes, and utilizes primer5 .0 and other molecular biology software analyzed and compared the collected sequences, screened out the specific conserved sequences of the above genes and designed primers SEQ ID NO: 1-12; on the premise of ensuring the specificity of the primers and the absence of dimer effects, six breeds of pigs were bred Barrier viruses are divided into two groups of multiplex PCR systems (the strains are preserved by our laboratory, see Table 1 for details), the first group is PPV, PCV 2 and PRV, see figure 2 ; the second group is CSFV, PRRS and JEV, see image 3; Add a TAG sequence to the 5' end of the specific upstream primer, use 12 carbon bridges as a t...
Embodiment 2
[0035] Embodiment 2 kit composition
[0036] Multiplex PCR reagent: PCR system 25μL
[0037] Combination 1 (including 12.5 μL of multiplex PCR Mix, 0.5 μL of primers for PPV, PCV-2 and PRV, supplemented with ddH2O to 25 μL) (such as SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 3, SEQ ID NO: 9), combination 2 (comprising 12.5 μL of multiplex PCR Mix, 0.5 μL of primers for CSFV, PRRSV and JEV (such as SEQ ID NO: 4, SEQ ID NO :10, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:6, SEQ ID NO:12), reverse transcriptase 1μL, ddH2O supplemented to 25μL)
[0038] Liquid phase chip reagent: 90 μL of reaction system, including 1 μL of each coded reaction microsphere, 1×Tm supplemented to 20 μL (as shown in SEQ ID NO: 13-18), 70 μL of SAPE diluent (69.3 μL of 1×Tm and 0.7 μL of SAPE ).
Embodiment 3
[0039] Example 3 Kit usage method
[0040] 1. Nucleic acid extraction and multiplex PCR
[0041] For single PPV, PCV2, PRV, CSFV, PRRSV and JEV six virus cell cultures, PPV, PCV2, PRV, CSFV, PRRSV and JEV six virus mixtures (equal volumes of PPV, PCV2, PRV, CSFV, PRRSV and Mixture of JEV cell culture), and the nucleic acid of unknown virus in the supernatant of the treated clinical sample was extracted by MagPure Viral RNA Kit forMagMax ExPRVess nucleic acid extraction kit (Meiji Company). Spectrophotometer to measure its concentration. For the sample, add 5 μL proteinase K solution, 5 μL magnetic beads, 90 μL lysate and 50 μL sample or control to well A of a 96-well plate; add 150 μL washing solution to well B, and add 150 μL desalting solution to well C and D , add 50 μL sterilized deionized water to well H; put the 96-well plate with samples and reagents and the elution strip into the instrument, and start the program; for detection, or store at -70°C.
[0042] The reac...
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