High throughput test method for tomato bacterial disease by locking-type probe

Abstract

The invention relates to a high throughput test method for a tomato bacterial disease, and more particularly relates to a high throughput test method which can utilize a locking probe to carry out reverse dot blot hybridization on tomato bacterial wilt original bacteria, tomato ulcer pathogenic bacteria and a tomato spot disease, and belongs to the crop preventing and curing disease and exit and entry plant quarantine range. The high throughput test method provided by the invention is based on a rolling circle amplification technology of the locking probe, a pair of universal primers are utilized for amplifying multiple pathogens, then, the high throughput test method and a reverse dot blot are combined together, a result is judged through a chromogenic reaction, the testing time of the traditional tomato bacterial disease is shortened, the specificity and the sensitivity of the detection are guaranteed, and the high throughput test is realized. According to the method, the testing cycle of the traditional pathogenic bacteria can be shortened, the cargo clearance can be increased, the intercept and capture relevance ratio of epidemic situations of imported bulk farm-product is improved, the pest invasion capacity is improved, the cargo clearance of cargoes imported and exported at ports in China is accelerated, and the enterprise cost is reduced and the high throughput test method has the important meanings.

Description

technical field [0001] The present invention relates to a high-throughput detection method for tomato bacterial diseases, and more particularly to a high-throughput detection method for reverse dot hybridization of tomato bacterial wilt pathogen, tomato canker pathogen and tomato spot disease using padlock probes , belonging to the category of crop disease prevention and treatment and entry-exit plant quarantine. Background technique [0002] Tomato bacterial canker, tomato bacterial spot and tomato bacterial wilt are three important diseases in tomato production. The pathogenic bacteria responsible for these diseases are Clavibacter michiganensis subsp. michiganensis (hereinafter referred to as CMM), Pseudomonas syringae .pv. Tomatoes (hereinafter referred to as PST), Ralstonia solanacearum Race1 (hereinafter referred to as RS1). These diseases mainly affect tomato economic crops, causing serious economic losses, and are the most difficult bacterial diseases of t...

AI Technical Summary

Problems solved by technology

Traditional methods for detecting and identifying pathogens include pathogen isolation, purification, biochemical detection, serological identification, and pathogenicity detection[4], etc., but these methods have low sensitivity and lack of specificity. Long time [5], and it is difficult to detect a large number of samples in a short time

In addition, there are many other identification methods, but there are also certain defects.

such as identification P. syringae .pv. Tomatoes method of radioactive hybridization of bacterial clones in semiselective media, but it is limited by the toxins produced by the strains [6]

Conventional PCR cannot perform accurate quantitative analysis [7] , although Real-time PCR can quantitatively detect many pathogenic microorganisms and viruses [8-9] , but there are often false positive results

Moreover, all these methods cannot achieve the purpose of detecting multiple diseases in the same system.

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