Multiplex fluorescence immunoassay primer, kit and method for rapidly distinguishing four respiratory pathogens
A multiple fluorescence and immunoassay technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of higher requirements and increased difficulty of experiments, and achieve lower detection costs, less sample consumption, Guaranteed the effect of annealing temperature and hybridization efficiency
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Embodiment 1
[0089] Example 1 Primers
[0090] After screening a large number of designed primers, it was found that the primer pairs N1 and N2, A1 and A2, B1 and B2, L1 and L2 can quickly distinguish four kinds of avian respiratory pathogens simultaneously (chicken Newcastle disease virus, avian influenza virus, chicken infectious bronchitis virus and avian infectious laryngotracheitis virus), the base sequences of which are shown below.
[0091] N1: 5'-CTCATCTCAGACAGGGTCAATC-3' (SEQ ID NO: 1),
[0092] N2: 5'-ATGGAGTCACCAAGGGG-3' (SEQ ID NO: 2);
[0093] A1: 5'-ATGAGTCTTCTACCCGAGG-3' (SEQ ID NO: 3),
[0094] A2: 5'-TCAGAGGTGACAGGATTG-3' (SEQ ID NO: 4);
[0095] B1: 5'-AATCACGCTCAAGTTCAAGGC-3' (SEQ ID NO: 5),
[0096] B2: 5'-GCATTGTTCCTCTCTCTCATC-3' (SEQ ID NO: 6);
[0097] L1: 5'-GCAGCGAAAAGAAGGC-3' (SEQ ID NO: 7),
[0098] L2: 5'-TCATCGTGCTCGGTGTCC-3' (SEQ ID NO: 8).
[0099] The present invention adopts the method of multiple fluorescence immunoassay to distinguish four kinds of...
Embodiment 2
[0105] Example 2 The kit for multiple fluorescence immunoassays for rapidly distinguishing 4 kinds of poultry respiratory pathogens
[0106] The kit includes the following components:
[0107] (1) The primers designed in Example 1 for multiplex fluorescent immunoassay;
[0108] (2) 4 kinds of fluorescently encoded microspheres containing anti-tag sequences with different fluorescent colors, and the anti-tag sequences can be complementary to the tag sequences in the multiple fluorescent immunoassay primers; all 4 kinds of microspheres can be purchased From luminex company, the number of fluorescently encoded microspheres corresponding to Newcastle disease virus (NDV), avian influenza virus (AIV), chicken infectious bronchitis virus (IBV) and chicken infectious laryngotracheitis virus (ILTV) respectively is MTAG-A034 , MTAG-A029, MTAG-A036 and MTAG-A067.
[0109] (3) Streptavidin-phycoerythrin complex.
Embodiment 3
[0110] Example 3 Establishment of Multiple Fluorescence Immunoassay Detection Method for 4 Kinds of Poultry Respiratory Tract Pathogens
[0111] (1) Construction of four avian respiratory pathogenic plasmids
[0112] The RNA of NDV, AIV, IBV, and ILTV pathogens were extracted with Tiangen’s automatic nucleic acid extractor, and the primer pairs N1 and N2, A1 and A2, B1 and B2, L1 and L2 were used for RT-PCR amplification, and the amplified The products were detected by agarose gel electrophoresis and purified by gel cutting. The purified cDNAs were respectively connected to the pMD-19T vector, and the ligated products were transformed into DH5a competent cells. Single clones were selected and identified by colony PCR. Colonies of positive bacteria were subjected to plasmid extraction and sent for sequencing.
[0113] (2) Plasmid PCR amplification
[0114] The primers described in Example 1 were used to amplify NDV, AIV, IBV, and ILTV single, double, and quadruple PCR respect...
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