Primers, kits and method for fluorescent quantitative PCR detection of bursaphelenchus xylophilus or bursaphelenchus mucronatus

Abstract

The invention discloses primers, kits and a method for fluorescent quantitative PCR detection of bursaphelenchus xylophilus or bursaphelenchus mucronatus, wherein the fluorescent quantitative PCR detection primer for bursaphelenchus xylophilus comprises a PCR amplification primer I; the PCR amplification primer I comprises a PCR amplification forward primer I with the nucleotide sequence shown inSEQ ID NO: 1 (5'-GTTCCGCCTACTGATGGTTC-3') and a PCR amplification reverse primer I with the nucleotide sequence shown in SEQ ID NO: 2 (5'-CGGCTTTTCATCATCCTACGC-3'); the fluorescent quantitative PCR detection primer for bursaphelenchus mucronatus comprises a PCR amplification primer II; and the PCR amplification primer II comprises a PCR amplification forward primer II with the nucleotide sequenceshown as SEQ ID NO: 4 (5'-TTCTCTACGCAGCACGTTTGTCCG-3') and a PCR amplification reserve primer II with the nucleotide sequence shown as SEQ ID NO: 5 (5'-TTCACGAGACAGAGCTCACAACAAA-3'). The invention hasthe advantages of good DNA extraction effect, simple operation, short time consuming (within 1 hour), accurate and reliable results, and at least a single nematode can be detected, with the characteristics of high sensitivity and high specificity.

Description

Technical field [0001] The present invention relates to the field of forestry insect detection, and more particularly to fluorescence quantitative PCR detection primers, kits and methods of pine or pose nematodes. Background technique [0002] Pine material nematode and proposal linearworms are a perforated animal door, a nematode, pad blade, a pulleys, an umbrella, is a pests that endanger pine trees. Pine wood nematode disease is also known as loose sickness, which is spread through the media insects such as Moque Tians. Pine trees in the pine body. Pine trees after the pinem nematode and the pose nematode infection, the coniferous yellowish brown or red brown, wilt, the resin secretion, the trunk can observe the invading hole or spawning of the cow, the ovulation of the ovate, the disease tree is dry, and finally rot. Therefore, nematode detection is an important basis for the prevention and control of forestry. At present, the detection method used in the laboratory is a simp...

AI Technical Summary

Problems solved by technology

The current detection method used in the laboratory is simple morphological identification after nematode isolation, but the process of isolating nematodes is relatively long, non-adult worms cannot be identified through morphological identification, and need to be cultivated to adult worms to be identified, and the results of morphological identification are also Rely on the experience of the inspector

At present, there are also cases of detection by ordinary PCR, but the detection method is relatively complicated and time-consuming, coupled with the unreasonable design of primers, the application is not widespread

In addition, CN104673926 discloses a fluorescent quantitative PCR detection technology, which expands the detection technology of pine wood nematode to a certain extent, but there are still problems such as inability to accurately detect pine wood nematode, long detection time, large errors in cotton swab sampling and general sensitivity

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