Multi-RPA primer and probe for simultaneously detecting NDV, IBV, H9N2-subtype AIV and detection method of multi-RPA primer and probe
A probe and multiplex technology, applied in the field of molecular biology detection, can solve the problems of taking a long time, difficult to identify clinical diagnosis, unable to provide timely guidance for epidemic diseases, etc., to achieve low cost, high sensitivity and strong specificity. Effect
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Embodiment 1
[0055] Example 1: Synthesis of primers and probes for detection of Newcastle disease virus
[0056] According to the comparison results of the Newcastle disease virus hemagglutinin neuraminidase (HN) gene sequence published in GenBank, select the sequence at the 587bp-621bp position in the nucleotide sequence of the Newcastle disease virus HN gene (5'-GCAGAGATCACTCACACTCACATCAGTATTTAGCAC-3') As the upstream primer of the RPA amplification reaction; the reverse complementary sequence of the 693bp-728bp position sequence in the nucleotide sequence of the Newcastle disease virus HN gene is used as the downstream primer of the RPA amplification reaction, and the 5' end of the downstream primer is used to label the organism when synthesizing the primer Biotin:
[0057] 5'-Biotin-GTTGCACTCACACTGCAAGACTTCCGATTTTGGGTG-3'.
[0058] Using the sequence at the 634bp-681bp position in the nucleotide sequence of the Newcastle disease virus HN gene as a probe, when synthesizing the probe, m...
Embodiment 2
[0060] Example 2: Synthesis of primers and probes for detection of infectious bronchitis virus
[0061]Since there are many serotypes of chicken infectious bronchitis virus strains, the present invention selects the conserved nucleus of each serotype chicken infectious bronchitis virus strain genome on the basis of comparing the genome sequences of chicken infectious bronchitis virus strains of each serotype. Nucleotide sequence, the design of primers and probes based on this conserved nucleotide sequence. Among them, the sequence at the 57bp-92bp position in the conserved nucleotide sequence (5'-CTTAACAAAACGGACTTAAATACCTACAGCTGGTCC-3') is used as the upstream primer; the reverse complementary sequence of the 204bp-242bp position sequence in the conserved nucleotide sequence is used as the downstream primer to synthesize For the downstream primer, label the 5' end of the downstream primer with biotin: 5'-Biotin-CTAGGTAGCCCAAGCAACGGATGTATAAAGTGAGACAC-3'.
[0062] Use the nucle...
Embodiment 3
[0064] Embodiment 3: The synthesis of primers and probes for detecting H9N2 subtype avian influenza virus
[0065] According to the comparison results of the M gene nucleotide sequences of the H9N2 subtype avian influenza virus published in GenBank, select the sequence at the 223bp-262bp position in the H9N2 subtype avian influenza virus M gene nucleotide sequence (5'-CACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAG-3') As an upstream primer; the reverse complementary sequence of the 552bp-558bp position sequence in the M gene nucleotide sequence is used as a downstream primer, and the 5' end of the downstream primer is labeled with biotin (Biotin) when synthesizing the primer: 5'-Biotin-CATGCCTGATTAGTGGGTTGGTGGTAGTCGCCATCTG- 3'; Use the 375bp-433bp position sequence in the M gene nucleotide sequence as a probe. When synthesizing the probe, label Digoxigenin at the 5' end of the probe sequence, and replace the base C at position 7082 with tetrahydrofuran (THF) , A 3-carbon spacer (Spac...
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