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Method for detecting bursaphelenchus xylophilus, detection primer and LAMP (loop-mediated isothermal amplification) detection kit of detection primer

A technology of pine wood nematodes and kits, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of cumbersome process, multiple samples, poor specificity, etc., and achieve wide application prospects and specificity Strong and stable effect

Active Publication Date: 2014-02-05
杭州益森键生物科技有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The morphological detection method has the following disadvantages: (1) Due to the influence of external factors such as host, environment, and geographical location, the morphology of B. xylophilus within and between species has changed greatly. The tip of the tail, the shape of the tail, and the shape of the male umbrella often change, which brings great difficulty to accurate identification.
(2) Since nematode morphological identification must be carried out by institutions with professional knowledge and equipment, the scope of application of morphological observation is greatly limited
The traditional method of extracting B. xylophilus from wood chips is the CTAB method. The main disadvantages of this method are: more samples are required, the extraction time is long, the process is cumbersome, it must be carried out in the laboratory, and it cannot be applied in the field environment, so it cannot meet the actual conditions. detection needs
The key to the successful detection of B. xylophilus by using the loop-mediated isothermal amplification technique is to design a LAMP primer set with strong specificity and high sensitivity for the target gene of B. xylophilus. The existing LAMP primer set for detecting B. xylophilus , there are defects of poor specificity and low sensitivity to varying degrees, which need to be improved

Method used

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  • Method for detecting bursaphelenchus xylophilus, detection primer and LAMP (loop-mediated isothermal amplification) detection kit of detection primer
  • Method for detecting bursaphelenchus xylophilus, detection primer and LAMP (loop-mediated isothermal amplification) detection kit of detection primer
  • Method for detecting bursaphelenchus xylophilus, detection primer and LAMP (loop-mediated isothermal amplification) detection kit of detection primer

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0054] Experimental example 1 Optimization experiment of the extraction method of genomic DNA of pine xylophilus

[0055] 1. Using Chelex-100 as the extraction buffer to extract nematode DNA from pine wood

[0056] 1. Experimental method

[0057] Select 6 samples of pine wood containing pine wood nematodes, weigh about 100mg of wood blocks (containing about 25 nematodes), put them into a 5ml centrifuge tube, add 5% (w / v) Chelex-100 1ml, and stir with the tip of the pipette. Mix well, place on ice for 5 minutes, invert and mix well, boil in water for 5 minutes, draw the supernatant and use ITS region-specific primers for PCR amplification, take pictures by electrophoresis, and test the stability of the extraction method; take healthy wood samples and 20 pine wood nematodes artificially Mix, use the same method to extract nematode DNA, DNA extraction solution 2 n Double serial dilution, PCR amplification, electrophoresis, photographing, and quality analysis of the DNA extract....

experiment example 2

[0070] Experimental Example 2 Screening experiment of primers for detection of pine xylophilus LAMP

[0071] 1. Main experimental materials and methods

[0072] 1. Biomaterials

[0073] All nematodes used in this experiment are listed in Table 1. The wood samples were selected from dead pine trees in the same year, and discs with a thickness of about 5 cm were cut from the base, middle and crown base of the felled trunk, put into sampling bags, and brought back to the laboratory. Select 10g of rings within the bark and 3-5cm outside the heartwood, split them into wood chips with a thickness of 1-2mm with an axe, and use 6g for the separation of the Bellman funnel. After 24 hours, count with a nematode counting dish, and repeat 3 times for each sample ,take the average. The remaining 4g was used for LAMP diagnosis.

[0074] 2. Extraction of nematode DNA from wood chips for LAMP reaction

[0075] Weigh about 100mg of wood block (containing about 25 nematodes), put it into a...

experiment example 3

[0174] Experimental example 3 Detection of pine xylophilus using No. 4 LAMP primer set

[0175] 1. Experimental materials and general methods

[0176] 1. The specific primers for the LAMP reaction are as follows:

[0177] 4F3: 5'---TGTAAACACCGTATAAAGGAATT---3'; (SEQ ID NO. 13)

[0178] 4B3: 5'---AAGCGGTCTAAGCGAAAC---3'; (SEQ ID NO.14)

[0179] 4FIP:

[0180] 5'---CATCCTTTGGTCGCTTTCTGAGTTTTAAAAATTTCACCACGTTCG---3'; (SEQ ID NO.15)

[0181] 4BIP: 5'---CCGATTGTCTAACTTCTGCTGCGCTTGTTCTCTGAGACCATA---3'; (SEQ ID NO. 16)

[0182] Loop primer 4LBA: 5'---TTCCGAAAAGCTTGGGTAAAATCCT---3'. (SEQ ID NO. 33)

[0183] 2. See Table 1 in Experimental Example 2 for pine xylophilus and pseudo-pine xylophilus.

[0184] 3. Extraction of nematode DNA from wood chips for LAMP reaction

[0185] Weigh 0.05g of wood blocks containing nematodes, put them into a 2ml centrifuge tube, add guanidine isothiocyanate extraction buffer (3M guanidine isothiocyanate, 50mM Tris-Cl pH8.0, 20mM EDTA pH8.0, 1% T...

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Abstract

The invention discloses a method for detecting bursaphelenchus xylophilus, a detection primer and a LAMP (loop-mediated isothermal amplification) detection kit of the detection primer. The method for detecting the bursaphelenchus xylophilus comprises the steps of (1) extracting a DNA (deoxyribonucleic acid) of the bursaphelenchus xylophilus in a pine wood sample; (2) constructing a LAMP reaction system for loop-mediated isothermal amplification; (3) judging whether the bursaphelenchus xylophilus exists in the pine wood sample according to the change of fluorescence or turdity of an amplified product. The invention also provides a method which is easy to operate, low in cost and high in stability and is used for extracting the DNA of the bursaphelenchus xylophilus from pine wood. The invention further provides a LAMP primer group with high specificity and high sensitivity for detection of the bursaphelenchus xylophilus. The invention also provides the LAMP detection kit for the bursaphelenchus xylophilus. The LAMP detection kit comprises the LAMP primer group, a reaction buffering solution, a DNA chain replacement enzyme and a fluorescent dye / specific probe. The detection kit disclosed by the invention has the advantages of high specificity, high sensitivity, convenience and quickness in detection and the like.

Description

technical field [0001] The invention relates to a kit for detecting plant nematodes, in particular to a method for detecting pine wood nematodes (Bursphelenchus xylophilus), special LAMP detection primers and a LAMP detection kit, belonging to the field of detection of pine wood nematodes. Background technique [0002] Pine wood nematode (Bursphelenchus xylophilus) disease, also known as pine wilt disease, can lead to the death of pine trees in a short time, and is a devastating disease on pine trees. The host of the disease includes dozens of Pinus species such as black pine, red pine and Masson pine, and also harms a few non-pine conifers (Zhu Kegong, Zhu Zhengchang, Yan Aojin. Epidemiology and research progress of pine wood nematode [c]. Pine wood nematode disease in China The prevalence and governance of [M]. Beijing: China Forestry Press, 1995.). In 1905, Japan reported for the first time that the disease caused large-scale death of pine trees in Nagasaki Prefecture. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 汪来发王曦茁苟大平田国忠朴春根
Owner 杭州益森键生物科技有限公司
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