Multiple fluorescence immunoassay primer, kit and method capable of fast distinguishing CAV, MDV, REV and IBDV
A multiple fluorescence and immunoassay technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve difficult problems, reduce detection costs, avoid cross-hybridization, and simple operation
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Embodiment 1
[0094] Example 1 Primer Design
[0095] After screening a large number of designed primers, it was found that the primer pairs C1 and C2, M1 and M2, R1 and R2, B1 and B2 had the best effect on multiple fluorescence simultaneous detection of CAV, MDV, REV, and IBDV. As follows.
[0096] Primer C1: 5'-CGACATCGGAGGAGACAG-3' (SEQ ID NO: 1),
[0097] Primer C2: 5'-GGAAGCGGATAGTCATAGTAGA-3' (SEQ ID NO: 2);
[0098] Primer M1: 5'-CCCATTCCCTCTTCTGCC-3' (SEQ ID NO: 3),
[0099] Primer M2: 5'-GCTGAGCGTAAACCGTC-3' (SEQ ID NO: 4);
[0100] Primer R1: 5'-GACTGCCTTGTGACTGCT-3' (SEQ ID NO: 5),
[0101] Primer R2: 5'-ACTCCCACTGTTGTCTAAATC-3' (SEQ ID NO: 6);
[0102] Primer B1: 5'-ATGCGGAGCCTTCTGA-3' (SEQ ID NO: 7),
[0103] Primer B2: 5'-ATTAGCCCTGACCCTGTG-3' (SEQ ID NO: 8).
[0104] The present invention adopts the method of multiple fluorescence analysis to distinguish CAV, MDV, REV, and IBDV, so the above primers are further modified to meet the corresponding operation requirements....
Embodiment 2
[0110] Example 2 Multiplex Fluorescence Immunoassay Kit for Rapidly Distinguishing Chicken Infectious Anemia Virus, Chicken Marek's Disease Virus, Avian Reticuloendotheliosis Virus and Infectious Bursal Virus
[0111] The kit includes the following components:
[0112] (1) The multiplex fluorescent immunoassay primer set designed in Example 1;
[0113] (2) Four kinds of fluorescence-encoded microspheres containing anti-tag sequences encoding different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in multiple fluorescence analysis primers; luminex company, where CAV, MDV, REV, and IBDV correspond to the fluorescently encoded microsphere numbers MTAG-A019, MTAG-A065, MTAG-A056, and MTAG-015, respectively.
[0114] (3) Streptavidin-phycoerythrin complex.
Embodiment 3
[0115] Example 3 Establishment of multiple fluorescent immunoassay detection method for rapidly distinguishing chicken infectious anemia virus, chicken Marek's disease virus, avian reticuloendotheliosis virus and infectious bursal virus
[0116](1) Construction of CAV, MDV, REV and IBDV plasmids
[0117] Extract the nucleic acid (RNA / DNA) of CAV, MDV, REV, and IBDV pathogens with Tiangen’s automatic nucleic acid extractor, and use the primer pairs C1 and C2, M1 and M2, R1 and R2, B1 and B2 respectively designed in Example 1 RT-PCR amplification was carried out, and the amplified products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNA was connected to the pMD-19T vector with a kit from TaKaRa Company, the connected product was transformed into DH5a competent cells, single clones were selected, colony PCR identification was carried out, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send for se...
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