Specific primer pair for detecting cyprinid herpesvirus 2, probe and detection kit
A detection kit and primer pair technology, applied in the field of probes, kits and primers, can solve the problems of limiting the use of LAMP method and false positive results.
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Embodiment 1
[0065] 1. Obtaining the positive plasmid of CyHV-2: the common CyHV-2 ORF71 gene listed in the NCBI search literature, using the Vector NTI software to compare and find out its conserved region, and select the ORF71 gene partial region as the target amplification segment, It was constructed into the vector puc57 to prepare a CyHV-2 positive plasmid. The plasmid was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The partial sequence of the selected ORF71 gene is as follows:
[0066] GTCAAGTGCGCCTCTTTCAACCTACCCTTTAGCGTCAGGTCCATAGAGGATCCAGAGTACAGCGAGTGTCTGGATATGGTTCAGCACAACGTTAGCACCGTACGTTTCCAAGAGATTATGCAGTCTCGGGTGAGGACTTGCGAAGAGTTTGATTTCTACACGCCTCGCATCATGCATCAGGACAACGCGGTCAGACAACTCAACGAGTCTTGTATGAAAAAGACTGTGGGCGCCGAACGGATCTTCAAGCCCAAGATCAATCACAATAACGTGCAGAACCCGGACGAGCGTAGAAAGTTTGCAGCCGTGGTCCGTCAACGGTTCAAGCACATTGACTTCTTTCAAGGCGTCCGAATCAAGGTCGGATCTCTGGTGTGCGTACTAAAATATCAAACTCAAGTGTTTGAAGGCTGTCTGGGAATAGTGGAATCAGTACAACCCGTCATGGTACGCCTTTTTTTGTTTGTTTGTTTGTTTGATGAGACGATGGTGACT...
Embodiment 2
[0093] Select the following primers and probe sequences:
[0094] The upstream primer sequence is: 5'-CGCCTCTTTCAACCTACCCTTTAGCGTCAG-3' (SEQ ID NO: 3);
[0095] The downstream primer sequence is: 5'-GTCCGGGTTCTGCACGTTATTGTGATTGAT-3' (SEQ ID NO: 4);
[0096] The probe sequence is: 5'-TGTTTGTTTGTTTGTTTGATGAGACGATGG(BHQ1-dT)G(THF)C(FAM-dT)ATGGTTAATGTGTTGT(C3-SPACER)-3'(SEQ ID NO: 9)
[0097] By synthesizing a plasmid containing the CyHV-2ORF71 gene sequence as the detection target, the method version amplification test of recombinase polymerase amplification (combined with endonuclease IV) was carried out, and the 50 μl amplification reaction system was constructed as follows:
[0098] 60mM tris-acetate buffer pH8.0
[0099] 100mM potassium acetate
[0100] 14mM magnesium acetate
[0101] 3mM Dithiothreitol
[0102] 5% polyethylene glycol (20000)
[0103] 2mM ATP
[0104] 20mM creatine phosphate
[0105] 100ng / μl creatine kinase
[0106] 600ng / μl E. coli SSB protein
[...
Embodiment 3
[0121] Select the primer pair and probe sequence designed in Example 2, utilize the recombinase polymerase amplification (in conjunction with endonuclease IV) method amplification reaction system to amplify, construct 50 μ l amplification reaction system as follows:
[0122] 60mM tris-acetate buffer pH8.0
[0123] 100mM potassium acetate
[0124] 14mM magnesium acetate
[0125] 3mM Dithiothreitol
[0126] 5% polyethylene glycol (molecular weight 20000)
[0127] 2mM ATP
[0128] 20mM creatine phosphate
[0129] 100ng / μl creatine kinase
[0130] 400ng / μl E. coli recA protein
[0131] 200ng / μl E. coli SSB protein
[0132] 60ng / μl E. coli recO protein
[0133] 40ng / μl E. coli recR protein
[0134] 60ng / μl E. coli recF protein
[0135] 8Units Bacillus subtilis DNA polymerase I
[0136] 50ng / μl Endonuclease IV
[0137] 450 μM dNTPs
[0138] 420nM per upstream primer
[0139] 420nM each downstream primer
[0140] 120nM fluorescent probe
[0141] The template is a synt...
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