Kit for detecting pine beam nematode and detecting process thereof

Abstract

The invention discloses a pine wood nematode detecting reagent box which takes 5'ACC ATT CGG TTG GCT CTG TT 3' as a forward primer (F), takes a 5' CCC TAA GGC GTC GGT GAA C 3' as a reverse primer (R) and takes 5' FAM- TAG CTG AGC ATC TTT T-TAMRA 3' as a fluorescent probe (P). The invention also discloses a method for adopting the reagent box to detect the pine wood nematode. The primers and the probe in the pine wood nematode detecting reagent box of the invention have higher specificity, sensitivity as well as stability and provides a fast, sensitive and specific method for detecting the pine wood nematode.

Description

technical field [0001] The invention belongs to the field of plant protection and quality inspection, and relates to a pine wood nematode detection kit and a detection method thereof, in particular to a pine wood nematode real-time fluorescent PCR detection kit and a detection method thereof. Background technique [0002] Pine wood nematode [Bursaphelenchu ​​xylophilus (Sleiner&Buhrer) Nickle], also known as pine wilt disease, is spread by Monochamus alternatus Hope and mainly causes pine tree wilting, which can lead to pine tree death in a short period of time. a devastating disease. Hosts include dozens of pinus species including black pine (Pinus thunbergii), red pine (P.densiflosa) and masson pine (P.massoniana), and also harm a few non-pine conifers. The disease was first reported in Japan in 1905, and it has been distributed in many countries such as the United States, Canada, Mexico, Portugal, South Korea, Japan, and China, among which the most severe cases occurred ...

AI Technical Summary

Problems solved by technology

Due to similar morphological characteristics, the overlapping phenomenon of B. xylophilus and other nematodes brings great difficulty to accurate identification.

Second, due to the rich variety of nematodes in nature and the small size of nematodes, professional technical knowledge and skills are required to accurately identify a certain nematode, so in the actual detection process, there are often false detections and missed detections

Third, some pine logs and wood packaging materials intercepted by various quarantine inspection stations and quarantine ports have less water content and less nematode content, and most of them are larvae, so it is difficult to accurately identify them morphologically.

[0005] At present, in the molecular detection of pine wood nematode, mainly can be divided into two classes, a class is the method of traditional PCR, for example, the patent " pine wood nematode detection kit and detection method thereof " of Nanjing Agricultural University (application number: 200310106109.5 ), Yunnan Agricultural University's patent "a rapid detection method for pine wilt disease pathogenic nematode" (application number: 200510048722.5) and Ye Jianren's patent "Pine Xylophilus PCR detection specific primer pair" (application number: 200610039049.3), the above The methods all have the characteristics of requiring many instruments and equipment, complex sample processing, too many experimental links, difficult to master, and easy to introduce human errors.

The other is the method of real-time fluorescent PCR, such as the patent "a method for detecting pine wood nematode and its special primers and probes" (application number: 200310109374.9) of the Institute of Microbiology, Chinese Academy of Sciences, and the prevention and quarantine of forestry diseases and insect pests in Shanghai. Station and Nanjing Agricultural University Plant Protection Institute's patent "Pine wood nematode detection kit and its detection method" (application number: 200510026710.2), since the primers and probes used in the above detection method all come from ribosomal rDNA and microsatellite DNA, these DNA sequences are extremely conservative, which may lead to insufficient specificity and stability of primers

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