CN-121975981-A - Specific primer pair, primer probe combination, kit and method for detecting white spot syndrome virus

Abstract

The invention relates to the technical field of PCR detection, in particular to a specific primer pair, a primer probe combination and a kit for detecting white spot syndrome virus, wherein the primer probe combination comprises a specific primer and a probe aiming at white spot syndrome virus, the primer and the probe have good specificity, sensitivity and repeatability, and the rapid and accurate identification of white spot syndrome virus can be realized by combining an optimized real-time fluorescence PCR detection method. The kit can realize high-sensitivity detection of white spot syndrome virus, is suitable for early diagnosis of low-load virus, has stable and reliable detection results, can meet the standardized detection requirement of a laboratory, has simple and convenient operation and short detection period, and is suitable for rapid screening application of a basic laboratory. The invention provides a high-efficiency and practical detection technical support for early warning of white spot syndrome virus, epidemic situation prevention and control and healthy development of shrimp and crab breeding industry, and has good popularization and application prospect.

Inventors

• WANG NANNAN
• YAO HONGLIANG
• LI XINGYAN
• CHEN TAO
• Shen Guanglong
• Du Hechao
• MAO QING
• ZHANG LEI
• ZHOU ZIYANG
• GUO YUFEI

Assignees

• 金陵科技学院

Dates

Publication Date

20260505

Application Date

20260330

Claims (10)

1. 1. A specific primer probe combination for detecting white spot syndrome virus is characterized in that, Comprises an upstream primer, a downstream primer and a probe; The nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3.
2. 2. A specific primer probe combination for detecting white spot syndrome virus according to claim 1, The 5 'end of the probe is connected with a fluorescence report group, and the 3' end of the probe is connected with a fluorescence quenching group; The fluorescence reporter group is 6-FAM and the fluorescence quenching group is BHQ1.
3. 3. Use of a specific primer probe combination according to claim 1 or 2 for the preparation of a white spot syndrome virus detection product.
4. 4. A kit for detecting white spot syndrome virus comprising the primer probe combination of claim 1 or 2.
5. 5. The kit according to claim 4, wherein, The kit also comprises a premix, a positive standard and a negative standard.
6. 6. The kit according to claim 5, wherein, The positive standard is recombinant plasmid pUC-Kan-vp24 containing white spot syndrome virus gene fragment, the plasmid copy number concentration is 0.4X10 5 copies/. Mu.L, and the negative standard is DEPC water.
7. 7. A method for detecting white spot syndrome virus for non-diagnostic purposes, characterized in that, Detecting the sample by the kit according to any one of claims 4-6, and judging whether the sample has white spot syndrome virus or not according to the fluorescent signal.
8. 8. The method for detecting white spot syndrome virus according to claim 7, The fluorescence quantitative PCR detection reaction system of the kit comprises 10 mu L of 2X TAQMAN FAST QPCR MASTER Mix, 0.7 mu L of 10 mu mol/L of upstream primer, 0.7 mu L of 10 mu mol/L of downstream primer, 0.4 mu L of 10 mu mol/L of probe, 1 mu L of template, 8.2 mu L of DEPC water and 20 mu L of total volume; The reaction procedure was 94℃pre-denaturation for 3 min, 94℃denaturation for 5s, 60℃annealing for 30 s,40 cycles.
9. 9. A specific primer pair for detecting white spot syndrome virus is characterized in that, The primer comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
10. 10. Use of a specific primer pair according to claim 9 for the preparation of a white spot syndrome virus detection product.

Description

Specific primer pair, primer probe combination, kit and method for detecting white spot syndrome virus Technical Field The invention belongs to the technical field of biology, and particularly relates to a specific primer pair, a primer probe combination, a kit and a method for detecting white spot syndrome virus. Background White spot syndrome virus (White spot syndrome virus, WSSV) is a pathogen that primarily infects crustaceans such as shrimp and crab, and white spot syndrome caused by it is a very serious infectious disease. The disease is characterized by white spots on the chest nails of the ill shrimps and crabs, and is rapidly outbreaks in a plurality of countries and regions worldwide since the first discovery in southeast Asia in early 1990 s, which causes serious economic loss to the aquaculture industry. WSSV belongs to the genus Leptoviridae (NIMAVIRIDAE) white spot virus (Whispovirus), is a double-stranded circular DNA virus, has a genome size of about 300 kb, and is the largest known genome virus at present. The whole virus particle is in a rod shape, and has the size of about 80-120 nm multiplied by 250-380 nm, and is sequentially in a three-layer structure of a capsule membrane, a capsid and a nucleus pulposus from outside to inside. The virus has wide host range, and can infect various sea water, freshwater shrimp, crab crustacean and aquatic insect larvae. The transmission paths mainly comprise horizontal transmission (such as feeding an intermediate host carrying viruses or sick and dead shrimps) and vertical transmission (via eggs). The WSSV has a certain incubation period after infection, and is easy to induce disease outbreak when the environmental conditions are worsened, such as the changes of physical and chemical factors of salinity, temperature, ammonia nitrogen concentration and the like. Viruses proliferate in large quantities in a host body mainly by invading tissues such as gills, livers and pancreas of shrimps and crabs, interfere with replication, transcription and translation of genetic materials of the host cells, and finally lead to death of the host cells. In terms of detection technology, with the development of molecular biology technology, the WSSV detection method has gradually evolved from traditional pathological observation to nucleic acid detection technology. Currently, polymerase Chain Reaction (PCR) is the most widely used detection means. However, the conventional PCR detection has the limitations of low sensitivity, long detection time, and the need of judging the result by agarose gel electrophoresis. Although loop-mediated isothermal amplification (LAMP) is used for WSSV detection, the method requires three pairs of primers, the primers have complex design and are easy to interfere with each other, the detection stability is affected, and the method is not beneficial to subsequent sequencing identification. In spite of the combination of various technical advantages of the DETECTR detection system developed in recent years, two sets of primers still need to be designed, and the uncapping operation is easy to cause aerosol pollution, so that false positive results are caused. In view of the above, the existing WSSV detection methods still have shortcomings in terms of sensitivity, stability, operation convenience and the like, and development of a technical method which is simple and convenient to operate, high in sensitivity, good in stability and suitable for rapid screening in a basic laboratory is needed. Therefore, a rapid, accurate and high-sensitivity WSSV detection technology is established, and the method has important practical significance for early diagnosis, epidemic prevention and control and sustainable development of the breeding industry of crustacean white spot syndrome such as shrimps, crabs and the like. Disclosure of Invention In order to overcome the problems in the prior art, the invention provides a specific primer pair, a primer probe combination, a kit and a method for detecting white spot syndrome virus, which can realize high-specificity and high-sensitivity detection of white spot syndrome virus and are suitable for early diagnosis of low-load viruses. In order to achieve the above purpose, the present invention adopts the following technical scheme: The invention provides a specific primer probe combination for detecting white spot syndrome virus, which comprises an upstream primer, a downstream primer and a probe, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the probe is shown as SEQ ID NO. 3. Further, the 5 'end of the probe is connected with a fluorescence report group, the 3' end of the probe is connected with a fluorescence quenching group, the fluorescence report group is 6-FAM, and the fluorescence quenching group is BHQ1. The invention also provides application of the specific p