Method for identifying bentgrass nematode by real time fluorescent PCR technology

Abstract

The invention discloses a method for identifying Anguina agrostis by applying a real-time fluorescent PCR technology. The specific DNA sequence of the Anguina agrostis is utilized; a bioinformatics method is used to design a specific primer and a specific probe; a 5' end of the probe is labeled with a FAM; a 3' end of the probe is labeled with TAMRA; a target gene of the Anguina agrostis is amplified by applying a fluorescent PCR method; and whether the Anguina agrostis is contained in a sample is identified through the change of a fluorescent signal of the probe during the reaction. The method is not affected by larva instar and the quantity of polypides, is quicker and more accurate compared with morphology and morphometry identification methods, has simple operation, can be standardized, solves the problem of a standard method for quickly identifying the Anguina agrostis, greatly saves detection time, accelerates the acceptance of grass seeds at ports, promotes the import and export trade, and saves the cost generated by the long-term storage of goods in a goods yard.

Description

technical field [0001] The invention relates to a biological identification technology, in particular to a nematode molecular biological identification technology, that is, the technology of using real-time fluorescent PCR reaction to identify the nematode nematodes, especially the nematode nematodes bentgrass. Background technique [0002] Anguinaagrostis is an important species in the order Tylenchida, the suborder Tylenchina, the superfamily Tylenchoidea, the family Anguinidae, and the genus Anguina. Plant nematodes are important pests of pastures. Its host plants mainly include Agrostisspp., Calamagrostis spp., Carex spp., Dactylis spp., and Eragrostis spp. , Festuca spp., Hordeum spp., Lolium spp., Phleum spp., Poa spp., Puccinelliaspp .), Sporobolus spp., Trisetum spp., etc., mainly damage the inflorescence of the host plant, resulting in a decrease in the yield of seeds and shoots of the diseased plant. Especially in areas where forage such as bentgrass is widely p...

AI Technical Summary

Problems solved by technology

[0004] The identification of bentgrain nematodes is an important task in my country's entry-exit inspection and quarantine. However, there is no standard method for the identification of bentgrain nematodes in my country. The traditional morphological and morphometric identification methods are mainly based on the morphology of adult nematodes. It takes a long time to identify the biological characteristics and shape measurement data; and only when adults are found, combined with factors such as host plants and distribution areas, can be accurately identified. larvae, making accurate identification of bentgrass nematodes very difficult

Although the PCR amplification sequencing method studied in China and the PCR-RFLP method and multiplex PCR method studied in this laboratory can also accurately identify the nematode cnitting nematodes, the above two methods must have PCR amplification, gel electrophoresis and Gel imaging process, the procedure is complicated, and the identification time is long

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