PCR-RFLP method for identifying mucronate form of Bursaphelenchus xylophilus and round-tailed form of Bursaphelenchus xylophilus

Abstract

The invention discloses a PCR-RFLP (polymerase chain reaction- restriction fragment length polymorphism) method for identifying a mucronate form of Bursaphelenchus xylophilus and a round-tailed form of Bursaphelenchus xylophilus. The method comprises the following steps: DNA extraction, PCR amplification, reaction with restriction endonucleases HhaI and Hpy188I and spectral identification. If themaximum band of the PCR-RFLP spectrum resulting from the reaction with the restriction endonuclease HhaI is 467 bp, the mucronate form of Bursaphelenchus xylophilus is identified; if the maximum bandis 357 bp or 358 bp, the reaction with restriction endonuclease Hpy188I is carried out; and if the maximum band is 492 bp, the mucronate form of Bursaphelenchus xylophilus is identified. If the maximum spectral band is 743 bp or 741 bp, the round-tailed form of Bursaphelenchus xylophilus is identified. Therefore, the PCR-RFLP method can clearly identify the mucronate form of Bursaphelenchus xylophilus and the round-tailed form of Bursaphelenchus xylophilus and provides a guarantee for quarantine identification and treatment.

Description

technical field [0001] The invention relates to a PCR-RFLP identification technology of pine wood nematode; in particular, it relates to a PCR-RFLP identification method of M-type and R-type strains of pine wood nematode. Background technique [0002] The pine wood nematode Bursaphelenchus xylophilus is an important dangerous nematode that is concerned by countries all over the world. It is the "number one killer" of pine forests. At present, China, South Korea, the European Union, Australia, New Zealand and many other countries have listed pine wood nematode as a quarantine object. The B. xylophilus B.mucronatus is very similar to B. xylophilus. The main morphological differences are: it is generally considered that the tail of the female is finger-shaped, and the tip of the tail is more than 3.5 μm long, and the female is broad and round. B. xylophilus nematodes with no tail spike or finger-like tail spike with a tail spike length of about 1 μm (not more than 2 μm). In 19...

AI Technical Summary

Problems solved by technology

The internal transcriptional spacer (ITS) sequence of nematode ribosomal DNA has a relatively fast evolution rate and has been used in the research on the classification and identification of nematode species and intraspecies. The PCR-RFLP identification of pine xylophilus and its related species is currently published using Rsa I , Hae III, Msp I, HinfI and Alu I, these five restriction endonucleases, although they can distinguish B. xylophilus and B. xylophilus, they cannot distinguish M-type and R-type strains of B. xylophilus

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