One-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic diseases

Abstract

The invention discloses a one-step multiple RT-PCR detection method for simultaneously detecting 3 pathogens of sugarcane mosaic diseases. Through specific primers on sugarcane streak mosaic viruses,sorghum mosaic viruses and sugarcane mosaic viruses and the optimizing of specific primer concentrations, enzyme dosage and annealing temperatures, the reverse transcription and PCR amplification of the 3 pathogens can be established in a same reaction tube through one-time RT-PCR reaction; the multiple RT-PCR detection method simultaneously accomplished in one step under a same reaction conditionis simple in operation step; repeated detection of conventional RT-PCR or complex two-step multiple RT-PCR reaction steps and programs, long time consumption and cross contamination can be avoided; and the method has the characteristics of being simple, efficient, strong in specificity and high in sensitivity, can be used for the production, application, germplasm exchange and introduction quarantine of sugarcane virus-free and healthy seedlings, and has important significance on preventing and controlling the spreading of sugarcane mosaic diseases.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, and specifically relates to a one-step multiplex RT-PCR detection method for simultaneously detecting sugarcane mosaic virus, sorghum mosaic virus and sugarcane mosaic virus three pathogens of sugarcane mosaic disease. Background technique [0002] Sugarcane is the most important sugar crop in my country, its sugar production accounts for more than 85% of the total sugar production in the country, and occupies an important position in the agricultural economic income. Sugarcane mosaic disease is a worldwide sugarcane "cancer" caused by a virus, which leads to poor quality of infected plants, decreased seedling germination rate, decreased tillering rate, decreased sucrose crystallization rate, and yield loss of 30% when the infection rate exceeds 30%. It has become one of the most widely distributed and most harmful diseases in my country's sugarcane areas, causing hundreds of million...

AI Technical Summary

Problems solved by technology

At present, the genetic detection methods of sugarcane virus mainly include PCR, RT-PCR and ring-mediated isothermal amplification technology, among which conventional RT-PCR is currently the most widely used RNA virus detection method, but conventional RT-PCR can only detect a single RNA virus at a time. virus detection

For the sugarcane mosaic disease that often occurs in the field with two or more pathogenic co-infections, using conventional RT-PCR for pathogen detection requires 2 or 3 RT-PCR, which is cumbersome and requires reagents. There are many types and high cost. It takes at least 8 hours to get the result of testing a sample, which takes a long time and has low detection efficiency, which cannot meet the needs of modern production.

Existing multiplex RT-PCR detection mostly adopts a two-step method, which needs to complete the detection of pathogens in two reaction systems. The reaction steps and procedures are relatively complicated, time-consuming, and easily cause cross-contamination

[0005] With the application of detoxified and healthy sugarcane seedlings in sugarcane production, there is an urgent need for a more rapid, accurate, and high-throughput method for the detection of sugarcane mosaic disease pathogens. At present, there is no method for simultaneously detecting the three pathogens of sugarcane mosaic disease. Related reports on one-step multiplex RT-PCR detection

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