Molecular marker primer combination for rapidly and synchronously identifying Citrus huanglongbing, canker, Citrus tristeza virus, Citrus tatter leaf and citrus exocortis viroid, and method

Abstract

The invention discloses a molecular marker primer combination for rapidly and synchronously identifying Citrus huanglongbing, canker, Citrus tristeza virus, Citrus tatter leaf and citrus exocortis viroid and an identification method. According to the invention, a synchronous rapid extraction and one-step RTPCR reaction system for RNA and DNA of citrus leaf tissues is established, a multi-specificmolecular marker primer combination capable of synchronously identifying citrus yellow shoot, canker, Citrus tristeza virus, Citrus tatter leaf and citrus exocortis viroid is developed, and the primercombination is suitable for high-throughput typing detection platforms such as fluorescent capillary electrophoresis, is high in sensitivity, and can be used for rapid identification of citrus diseases. Based on the developed molecular marker primer combination, through repeated tests and optimization, the five common citrus diseases can be synchronously detected, important technical guidance significance is achieved for detecting large-scale citrus sample plate diseases, and the advantages of being easy to operate, efficient, rapid and the like are achieved.

Description

technical field [0001] The invention relates to a group of molecular marker primer combinations and a method for quickly and synchronously identifying citrus huanglongbing, canker, decay disease, broken leaf disease and split skin disease, and belongs to the field of molecular biology. Background technique [0002] Citrus Huanglongbing (HLB), Citrus Bacterial Canker Disease (CBCD), Citrus tristeza virus (CTV), Citrus Tatter Leaf Virus (CTLV) and Citrus cracking disease Virus (Citrus exocortis viroid, CEVd) is one of the five important diseases affecting citrus production, which seriously restricts the development of citrus industry in my country. The establishment of rapid and accurate detection methods is one of the most important prerequisites for the prevention and control of the spread of the above diseases. At present, some single rapid detection methods have been established for the above-mentioned pathogens, including serology, RT-PCR, nested PCR and detection methods ...

AI Technical Summary

Problems solved by technology

At present, some single rapid detection methods have been established for the above-mentioned pathogens, including serology, RT-PCR, nested PCR and detection methods based on loop-mediated isothermal nucleic acid amplification technology. It is widely used in the detection and identification of pathogens such as viruses; at the same time, some detection methods for pathogenic pathogens based on multiplex PCR technology have also been constructed, but they cannot achieve large-span multiple types of pathogenic pathogens (such as viruses, bacteria and viroids, etc.) Simultaneous detection; Among them, due to the differences in the nucleic acid structure types of pathogenic pathogens, citrus virus pathogens (mostly nucleic acid is a single-stranded RNA structure) are mainly realized through RNA extraction, reverse transcription and RT-PCR operation technology, while citrus bacterial pathogens (Nucleic acids are mostly DNA structures) The purpose of detection can be achieved mainly through DNA extraction and direct PCR amplification. The above two types of pathogens usually need to be divided into two types for separate identification and analysis. The operation steps are relatively cumbersome, and usually need to use chloroform, etc. Toxic reagents take a long time, and the workload and detection cost are also high, which is not conducive to large-scale promotion and application

[0003] In general, the DNA and RNA of plant pathogenic viruses or bacteria are mainly obtained from infected plant tissues, which are also the raw materials for routine pathogen detection. The extraction technology is very mature, but most of them are extracted separately. There are few reports on the simultaneous extraction of DNA and RNA from tissues, and the related extraction kits are expensive and not suitable for large-scale application, and most of them use a two-step precipitation method, usually precipitating RNA first, and then precipitating DNA , toxic organic solvents such as chloroform are often used in the test process, the steps are cumbersome and time-consuming

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