PCR-RFLP method for distinguishing four types of pratylenchus

Abstract

The invention discloses a PCR-RFLP method for distinguishing four types of pratylenchus. A pair of common primers is designed based on the DNA sequence of the D2-D3 region of the 28S gene of nematode ribosome for PCR amplification by DNA extraction, then four types of restriction enzyme are utilized to carry out digestion and band spectrum identification so as to obviously distinguish P. coffeae, P. loosi, P. penetrans and P. vulnus. The PCR-RFLP method has stable results, simple operation, and wide application prospects in the fields of plant quarantine and plant protection.

Description

Technical field [0001] The present invention relates to the detection technology of Brachymeria nematodes, in particular to a PCR-RFLP method for distinguishing 4 species of Brachymeria nematodes. Background technique [0002] Brachyzoites ( Pratylenchus Filipjev, 1936) is an important type of migratory plant endoparasitic nematode, which can severely damage plant root tissue and cause root rot. At present, there are nearly 80 species of Nematodes of the genus Brachyma that have been found in the world. In 2007, my country included nematodes (non-Chinese species) of the genus Brachyura into the "List of Entry Phytosanitary Pests of the People's Republic of China". Coffea elegans P. coffeae Brachymys lucifera P. loosi Puncture P. penetrans Paratymida P. vulnus It is common in the world, and the frequency of interception at Chinese ports is very high. Although these 4 species of nematodes are not the subject of quarantine, accurate identification of them will help us to make...

AI Technical Summary

Problems solved by technology

To identify 4 nematodes, 4 pairs of primers need to be used to react separately, the operation is relatively complicated, and the specific primers may lose their specificity due to the differences in the intraspecific DNA sequences of Brachyphysis nematodes from different sources

The PCR-RFLP method is also a traditional method for the identification of nematodes, but the target PCR fragments used before are all ribosomal ITS regions. The disadvantage is that different researchers use different types of ITS primers, resulting in differences in the size of PCR product fragments , and at least 5 kinds of restriction endonucleases are required for the currently used methods

In addition, due to the rapid evolution rate of the ITS region, there are sequence polymorphisms in the repeat fragments of the ITS region of the same species of nematodes, which leads to inconsistent RFLP results of the same species of nematodes and affects the identification of nematode species

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