PCR (Polymerase Chain Reaction) primer and method for detecting cucumber green mottle mosaic virus (CGMMV)

Abstract

The invention discloses a primer and a method for detecting cucumber green mottle mosaic virus (CGMMV). The primer is specific in the CGMMV, has the characteristics of being good in specificity and high in rate of coverage on different CGMMV isolators, can be used for effectively detecting the different CGMMV isolators, is more accurate in detection result, and is more convenient since auxiliary primers are not needed in the detection. The detection method disclosed by the invention is applicable to the direct detection on the CGMMV in a great number of seeds of cucurbit plants, and maintains the advantages of rapidness, accuracy, high sensitivity and the like while lowering the detection cost and reducing the work load.

Description

technical field [0001] The invention relates to a PCR primer for detecting cucumber green mottle mosaic virus in plants and a method thereof, in particular to a PCR primer for specifically detecting cucumber green mottle mosaic virus in seeds of cucurbit plants and a detection method thereof. Background technique [0002] Cucumber green mottle mosaic virus ( Cucumber green mottle mosaic virus , CGMMV) is one of the important members of the genus Tobacco Mosaic Virus, which mainly infects Cucurbitaceae plants such as cucumber, watermelon, melon, pumpkin, etc. Since it was first reported in the UK in 1935, it has been widely distributed in many countries in Asia, Europe, and South America. countries and regions, causing serious economic losses to the production of cucurbit crops. CGMMV was first discovered in Gaizhou City, Liaoning Province in 2005, and the incidence area of ​​watermelon reached 333 hm 2 , of which 13 hm 2 No harvest. In 2006 and 2007, the virus was list...

AI Technical Summary

Problems solved by technology

But the present inventor finds some drawbacks that existing detection method exists in actual detection: the one, there is primer specificity when PCR amplification is not good, and primer is to the problems such as low coverage rate of different CGMMV isolates; At present, the existing detection methods generally require to sow seeds first, and when the seeds grow to the 3rd or 4th leaf stage, the total RNA is extracted for detection after the leaves are sorted and collected, or the total RNA of a single or several seeds is directly extracted for detection. This method of extracting total RNA has disadvantages such as time-consuming, high cost, and heavy workload, and is not suitable for the detection of large batches of seeds

Although there are researchers in the prior art to explore the PCR detection method, there are also shortcomings of insufficient sensitivity and incomplete coverage. For example, the invention title applied for by the Chinese Academy of Inspection and Quarantine on January 18, 2011 is "rapid detection of cucumber green mottled flower". Leaf Virus Specific Primer Pair and Its Application” patent application (application number 201110020249.5), in which the specific primer invented does not reach 100% coverage with the CGMV whole genome nucleotide sequence, that is, the forward primer: 5' –GAAGAGTCCAGTTCTGTTTC-3' found four CGMMV genome nucleotide sequences with only 85% coverage in NCBI through BLAST search; on July 07, 2009, the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China released the "People's Republic of China The specific primers used for RT-PCR detection described in the "Quarantine and Identification Method of Cucumber Green Mottled Mosaic Virus" in "Industry Standards for Entry-Exit Inspection and Quarantine" all have the nucleotide sequence of the whole genome of CGMMV and the forward primer or the reverse primer. Cases where primers do not reach 100% coverage

The PCR primers of the above prior art obviously cannot detect some CGMMV isolates, and there is a possibility of detection errors

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