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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.

Active Publication Date: 2020-12-25
江苏省渔业技术推广中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although LAMP increases the amplification efficiency, non-specific pairing between primers easily leads to false positive results, which limits the use of LAMP methods

Method used

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  • Specific primer pair for detecting cyprinid herpesvirus 2, probe and detection kit
  • Specific primer pair for detecting cyprinid herpesvirus 2, probe and detection kit
  • Specific primer pair for detecting cyprinid herpesvirus 2, probe and detection kit

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a specific primer pair for detecting cyprinid herpesvirus 2 (CyHV-2), and a probe and a detection kit which are used in cooperation with the primer pair. According to the invention, a conservative gene ORF71 in the CyHV-2 is used as a detection target; and the specific primer and probe combination is used through an isothermal amplification technology, so that the detectionconvenience and specificity of the CyHV-2 are improved, and simultaneously, the detection time is also greatly shortened. Compared with a PCR detection method, the method disclosed by the invention has the advantages that: a product electrophoresis verification process is omitted; a false positive result is avoided; and the detection accuracy is improved. Compared with qPCR (Quantitative Polymerase Chain Reaction), the method disclosed by the invention is simple, convenient and feasible, does not need complicated instruments and equipment, saves the cost, improves the detection efficiency, and simultaneously, is convenient to popularize and use in a large range. Compared with other isothermal amplification methods, the detection method disclosed by the invention has the advantages that: the required time is relatively short; and the detection accuracy is relatively high.

Description

technical field [0001] The invention relates to primers, probes and kits, in particular to specific primer pairs, probes and detection kits for detecting carp herpesvirus type II. Background technique [0002] Cyprinid herpesvirus 2, or CyHV-2 for short, mainly infects goldfish hematopoietic organs, so it is also called goldfish haematopoietic necrosis virus (Goldfish haematopoietic necrosis virus, GFHNV) or herpesvirus hematopoietic necrosis virus (Herpesvirus). - pesviral haematopoietic necrosis virus, HVHNV). The CyHV-2 nucleocapsid is hexagonal or spherical, with a diameter of 100-110nm, and the diameter of the oval virion with envelope is 175-200nm. The virus is highly pathogenic to goldfish and crucian carp, and the mortality rate can reach almost 100%. Virus infections have occurred in Japan, the United States, Australia, the United Kingdom, and China, causing a huge impact on the ornamental fish farming industry and causing economic losses. In recent years, crucia...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/705C12Q2531/119C12Q2521/507C12Q2522/101C12Q2537/1376C12Q2563/107
Inventor 袁锐刘肖汉巫爱军王晶晶刘训猛陈静吴亚锋于继彬
Owner 江苏省渔业技术推广中心
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