CN-121992154-A - Multiplex RT-PCR method and kit for detecting important blueberry viruses

Abstract

The invention belongs to the technical field of plant virus molecular detection, and particularly discloses a multiplex RT-PCR method and a kit for detecting important blueberry viruses, and particularly provides a blueberry virus multiplex RT-PCR detection primer, wherein the primer sequence is shown as SEQ ID NO. 1-12, the technical scheme provided by the invention has wide detection range, the method can be used for simultaneously detecting the blueberry scorch virus, the blueberry shock virus standard strain and variant strains thereof, tomato fruit ring spot virus, blueberry necrosis mottle virus and apple mosaic virus, solves the problem that the variant strains and different virus strains cannot be detected by the traditional ELISA method, covers the main blueberry viruses and key virus types of cross-border infection, and has strong specificity and high sensitivity.

Inventors

• DING MING
• LI TINGTING
• YIN YUEYAN
• SU XIAOXIA
• ZHAO LILING
• ZHONG JING
• LIN TAO
• Dong ru

Assignees

• 云南省农业科学院生物技术与种质资源研究所

Dates

Publication Date

20260508

Application Date

20260305

Claims (7)

1. 1. The blueberry virus multiplex RT-PCR detection primer is characterized in that the primer sequence is shown as SEQ ID NO. 1-12.
2. 2. Use of the primer according to claim 1 for the preparation of blueberry virus detection and/or diagnostic products.
3. 3. A blueberry virus detection kit, characterized in that the kit contains the primer of claim 1.
4. 4. Use of the kit of claim 3 in blueberry virus detection, wherein the viruses include BlScV, blShV and variants thereof, TZSV, BNRBV and ApMV.
5. 5. A multiplex RT-PCR method for detecting blueberry important viruses, characterized in that the primer according to claim 1 or the kit according to claim 3 is used for detection.
6. 6. The method according to claim 5, wherein the PCR amplification is performed by using the primer according to claim 1 after extracting RNA of the sample to be tested and obtaining cDNA by reverse transcription, and the presence or absence of virus is determined based on the size of the band of the PCR product.
7. 7. The method of claim 5 or 6, wherein the virus comprises BlScV, blShV and variants thereof, TZSV, BNRBV, and ApMV.

Description

Multiplex RT-PCR method and kit for detecting important blueberry viruses Technical Field The invention belongs to the technical field of plant virus molecular detection, and particularly relates to a multiplex RT-PCR method for simultaneously detecting multiple viruses of blueberries and a special kit. Background Blueberry is used as a high-value cash crop, is widely planted in the world, and the Yunnan blueberry industry is in the forefront of the country in area, yield and yield value, and has become an important strength in the global blueberry layout. However, viral infection is an important factor restricting the development of the blueberry industry, wherein the blueberry scorch virus (Blueberry scorch virus, blScV), the blueberry shock virus (Blueberry shock virus, blShV) and variants thereof, the blueberry necrosis ring spot virus (Blueberry necrotic ring blotch virus, BNRBV), the apple mosaic virus (Apple mosaic virus, apMV) are four main viruses which are the most serious harm to blueberry production, and in addition, the tomato fruit ring spot virus (Tomato zonate spot virus, TZSV) is used as a cross-infection virus, and in recent years, it has been found that blueberry plants can be infected, ring spots appear on fruits, the quality is reduced, and the loss risk of the blueberry industry is further aggravated. The plant infected by the blueberry focal spot virus cannot be recovered and must be thoroughly shoveled, the plant infected by the blueberry focal spot virus can be recovered gradually over time but can obviously influence the fruit yield and quality, the plant infected by the blueberry necrotic spot virus can generate mottled leaves and necrosis, the plant dies when serious, and the plant infected by the apple mosaic virus can generate symptoms such as flower leaves, mottled leaves and the like, so that the appearance and commodity value of fruits are influenced. Plants infected with tomato fruit ring spot virus can have systematic mottle, necrosis and other symptoms, and the plants die when serious. At present, the conventional detection method of the blueberry virus is mainly an enzyme-linked immunosorbent assay (ELISA), but the method has obvious limitations. In recent years, research shows that new variant strains of blueberry focal wilt virus and blueberry shock virus appear, tomato fruit ring spot virus also spans the risk of species transmission, and the traditional ELISA detection method can not effectively identify the variant strains and different strains, so that the detection result of part of blueberry plants showing obvious symptoms is negative, great uncertainty is brought to growers, and the risk of virus diffusion transmission is increased. Polymerase Chain Reaction (PCR) technology is widely used in the field of virus detection due to its high specificity and high sensitivity. The multiplex RT-PCR technology can amplify specific fragments of various target viruses simultaneously in the same reaction system, realizes synchronous detection of various viruses, and has the advantages of high efficiency, high speed and low cost. In the prior art, multiple RT-PCR detection methods for various viruses of crops such as tomatoes exist, but multiple detection technologies for main viruses (especially including variant strains) of blueberries and viruses infected by crossing are not reported yet. Therefore, a set of multiple RT-PCR detection schemes capable of accurately detecting multiple viruses simultaneously are designed, and the method has important significance for early warning, accurate prevention and control and health development of blueberry industry. Disclosure of Invention Aiming at the problems that the existing blueberry virus detection technology cannot effectively detect variant strains, different virus strains, low detection efficiency and the like, the invention provides a multiplex RT-PCR method and a kit for simultaneously detecting a plurality of main blueberry viruses, and the method and the kit realize rapid, accurate and synchronous detection of important viruses in the blueberry seedling production process. In order to achieve the above purpose, the invention adopts the following technical scheme: The invention provides a blueberry virus multiplex RT-PCR detection primer, the primer sequence of which is shown as SEQ ID NO. 1-12. Furthermore, the invention provides application of the primer in preparation of blueberry virus detection and/or diagnosis products. In one embodiment, the invention provides a blueberry virus detection kit, which contains the primer. Furthermore, the invention provides application of the kit in blueberry virus detection, wherein the viruses comprise BlScV, blShV and variant strains thereof, TZSV, BNRBNRBV and ApMV. Furthermore, the invention provides a multiplex RT-PCR method for detecting the blueberry important virus, which is to detect by adopting the primer or the kit. Further, the method of the invention is that after RNA o