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Primer composition for identifying pine wood nematodes, product using same, applications thereof, detection method and applications thereof

A primer composition, pine xylophilus technology, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve the problems of high cost, poor specificity, cumbersome operation, etc., and achieve low cost and accurate The effect of identifying and eliminating the source of infection

Inactive Publication Date: 2018-07-24
RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The first object of the present invention is to provide a primer composition for identifying pine xylophilus, the second object of the present invention is to provide a monitoring method for identifying pine wood nematode using the above primer composition, and the third object of the present invention is to provide the above primers The application of the composition in the preparation of products for identifying pine wood nematodes, the fourth object of the present invention is to provide a reagent for identifying pine wood nematodes, and the fifth object of the present invention is to provide a reagent for identifying pine wood nematodes The nematode kit, the sixth purpose of the present invention is to provide the above-mentioned primer composition or reagent or the application of the kit in identifying whether Pine massoniana is infected with B. xylophilus, and the seventh purpose of the present invention is to provide the above-mentioned primer composition or reagent Or the application of the kit in the identification of pine wood nematode, to alleviate the problems of cumbersome operation, high cost or poor specificity of the identification method of pine wood nematode in the prior art

Method used

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  • Primer composition for identifying pine wood nematodes, product using same, applications thereof, detection method and applications thereof
  • Primer composition for identifying pine wood nematodes, product using same, applications thereof, detection method and applications thereof
  • Primer composition for identifying pine wood nematodes, product using same, applications thereof, detection method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Method for Extracting Pine Xylophilus DNA from Pinus massoniana

[0054] Use the CTAB Plant Genomic DNA Rapid Extraction Kit for extraction, but some of the steps are improved, the specific steps are as follows:

[0055] (a) From the inoculation point of Pinus massoniana stems (not including the inoculation point), take samples in 2 cm sections, take about 0.8 g of stem segments, add liquid nitrogen in a mortar, and grind them into fine powder.

[0056] (b) Transfer the fine powder in (a) to a 1.5ml centrifuge tube, do not thaw, add 600μl 65°C preheated lysate PL (confirm that β-mercaptoethanol has been added to 2%), vigorously vortex and mix, Lysis is aided by gentle pipetting with a wide-bore pipette tip.

[0057] If tissue lysis is difficult, a gentle homogenization step for 10 seconds can be added as needed to aid in lysis.

[0058] (c) 65°C water bath for 20-60 min, invert the centrifuge tube to mix the sample several times during the water bath.

[00...

Embodiment 2

[0069] Example 2 Extraction method of isolated pine wood nematode DNA

[0070] from ddH 2 Pick 1-2 of the cleaned pine wood nematodes and put them into a 200 μL PCR tube (containing 8 μL ddH 2 O and 1 μL 10×PCR Buffer (Mg 2+ free)), place the PCR tube in liquid nitrogen for 1 min, heat at 85°C for 2 min, then add 1 μL of 1 mg / mL proteinase K to the PCR tube, heat at 56°C for 15 min, then heat at 95°C for 10 min, and obtain the DNA extraction solution directly as a PCR Amplified template.

Embodiment 3

[0071] Example 3 Identification of pine xylophilus in vivo or in vitro

[0072] (a) The primers used in the experiment are as follows:

[0073] The primer sequences of the first primer pair are:

[0074] Upstream primer-FP: 5'-GATTCGCGTCGTTCCGCCTA-3' (SEQ ID No.1);

[0075] Downstream primer-RP: 5'-GGAACAAACGCTCCGCTAAA-3' (SEQ ID No.2);

[0076] The primer sequences of the second primer pair are:

[0077] Upstream primer-FP: 5'-CGTGCTGTTGTTGTTGAGTTGGC-3' (SEQ ID No.3);

[0078] Downstream primer-RP: 5'-AGACGACTGTCACAACGTGC-3' (SEQ ID No. 4).

[0079] (b) Nested PCR amplification uses a 50 μL reaction system:

[0080] First round of PCR, using the first pair of primers:

[0081]

[0082]

[0083] For the second round of PCR, use the second primer pair:

[0084]

[0085] (c) Nested PCR amplification procedure:

[0086] The first round of PCR: 95°C, 2min. 94°C, 30s; 63°C-53°C fall, 30s; 72°C, 1min; 25 cycles. 72°C, 5min.

[0087] The second round of PCR: 95°C,...

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Abstract

The invention relates to the field of biological control and detection, and specifically provides a primer composition for identifying pine wood nematodes, a product using the same, applications thereof, a detection method and applications thereof. The provided primer composition comprises a first primer pair and a second primer pair. The provided detection method is simple, convenient and fast, does have any special requirement on equipment, can carry out detection by using two primer pairs, and has a low detection cost. High specificity of Nested PCR is utilized, after two rounds of PCR, pine wood nematodes can be identified efficiently; and accurate results of identification of pine wood nematodes directly sampled from monochamus alternates or Chinese red pines, isolated pine wood nematodes, adult pine wood nematodes, and young pine wood nematodes can be obtained rapidly. The provided product is a reagent or a kit, and thus even a common person can rapidly and accurately identify pine wood nematodes.

Description

technical field [0001] The invention relates to the field of biological control detection, in particular to a primer composition for identifying pine xylophilus, a product using the same, an application thereof, a detection method and the application thereof. Background technique [0002] Pine wood nematodes belong to the phylum Nematozoa, the orders of the order, the superfamily, the family of the family, and the genus of the genus. Pine wood nematode is one of the most harmful invasive alien species in my country. It causes pine wood nematode disease, and the plants can die after 40 days of infection. It only takes 3-5 years from the onset of the disease to the destruction of the entire pine forest. A devastating pest and disease known as forest "cancer". Pine wood nematode disease is mainly transmitted through monochamus monochamus, wood and wood packaging materials. After being infected by pine wood nematode, the needle leaves are yellowish brown or reddish brown, wilti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2549/119
Inventor 刘青华刘彬周志春
Owner RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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