Eva Green fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for detecting salmonid alphavirus and application thereof
A fluorescence quantification and kit technology, which is applied in the fields of fluorescence quantitative PCR primers, salmon beetle virus detection, and virus detection primers and kits, can solve problems such as enlargement, and achieve simple preparation, good repeatability and high reliability. Effect
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Embodiment 1
[0038] The design of six genotype-specific primers of embodiment 1 salmon betavirus
[0039] According to the salmon alphavirus (SAV) E2 gene sequence (JX163854) published in Genebank, compare the E2 gene sequences of 6 strains of different genotypes of SAV, and screen out a conserved sequence (9471-9668bp). When designing primers, pay attention to avoiding mutation points in the sequence. After repeated screening and verification, a pair of EvaGreen fluorescent quantitative PCR primers for detecting six different genotypes of salmon alphavirus were obtained:
[0040]Upstream primer (TS1): 5'-GCAACATTGCCGATCTGAGGTGTGG-3' (SEQ ID NO.2)
[0041] Downstream primer (TS2): 5'-CGTAACATGCAACGACAGCCAGTGC-3' (SEQ ID NO.3)
[0042] The E2 gene of six genotypes of salmon alpha virus was amplified, the fragment size was 197bp, and its nucleotide sequence was shown in SEQ ID NO.1.
Embodiment 2
[0043] Embodiment 2 detects the establishment of the Eva Green fluorescent quantitative PCR detection method of salmon alpha virus
[0044] 1. Preparation of salmon alpha virus E2 gene recombinant plasmid standard:
[0045] The total RNA of salmon alphavirus SAV 4640 was extracted according to the total RNA extraction instructions, followed by reverse transcription according to the instructions of the TOYOBO reverse transcription kit, and the cDNA obtained by reverse transcription was amplified by conventional PCR with primers for amplifying the full-length gene of salmon alphavirus E2. Increase, the primer sequence is:
[0046] Upstream primer sequence: PF:5'-AGCTTCTTGCCGTCACCACCT-3' (SEQ ID NO.4);
[0047] Downstream primer sequence: PR: 5'-GCGGCCGCTTGGTCCACAAGTAG-3' (SEQ ID NO.5)
[0048] The PCR product was detected by 1.0% agarose gel electrophoresis, and the full length of the amplified E2 was 1375bp. The target product was recovered with a gel recovery kit, the recove...
Embodiment 3
[0059] The characteristic evaluation of embodiment 3 salmon betavirus Eva Green fluorescent quantitative PCR detection method
[0060] 1. Sensitivity evaluation, that is, the minimum detection limit of real-time PCR.
[0061] The concentration of positive reference DNA stock solution is 1.5×10 10 copies / μl. First dilute its 10-fold gradient to 1.5×10 1 copies / μl, and then use the solution of each dilution as a template to amplify using the fluorescent quantitative PCR method established in Example 2 of the present invention, and determine the minimum amount of template that can be detected by this method. According to the test results, the minimum detectable amount of this fluorescent quantitative PCR method is 1.5×10 1 copies / μl, indicating that the detection method of the present invention has high sensitivity.
[0062] In order to compare the sensitivity of the Eva Green fluorescent quantitative PCR primers established by the present invention and the common RT-PCR prim...
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