PCR-RFLP method for distinguishing four types of pratylenchus

A short-bodied nematode and nematode technology, applied in the field of PCR-RFLP, can solve the problems of complicated operation, inconsistent results, loss of specificity of specific primers, etc., and achieve the effects of simple operation, stable results and wide application prospects.

Inactive Publication Date: 2017-12-01
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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Problems solved by technology

To identify 4 nematodes, 4 pairs of primers need to be used to react separately, the operation is relatively complicated, and the specific primers may lose their specificity due to the differences in the intraspecific DNA sequences of Brachyphysis nematodes from different sources
The PCR-RFLP method is also a traditional method for the identification of nematodes, but the target PCR fragments used before are all ribosomal ITS regions. The disadvantage is that different researchers use

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  • PCR-RFLP method for distinguishing four types of pratylenchus

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Embodiment

[0013] The PCR-RFLP method to distinguish 4 species of Brachytematodes, the specific steps are as follows:

[0014] 1. DNA extraction: add an appropriate amount of sterilized double distilled water on a sterilized clean glass slide, and use a sterilized 0 gauge needle

[0015] The worms of the genus Brachyma are picked in water droplets for washing, and then the worms are picked up with 10μl of double distilled water (ddH 2 O) Microcentrifuge the sterilized PCR tube and place it in an ultra-low temperature refrigerator -80℃ for 29~31 min. After taking it out, keep it at 85℃ for 5 min, and then immediately reduce the temperature of the PCR tube to 56℃. Constant temperature for 30 S, add 2μl of proteinase K solution (purchased from TaKaRa) and 10×EasyTaq buffer 5μl (purchased from TRANS) with a concentration of 1 mg / mL to the PCR tube. The PCR tube is first kept at 56°C for 1 h , Then keep it at 95℃ for 15 min to obtain the DNA extract, and store it at -20℃ for later use;

[0016] 2. ...

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Abstract

The invention discloses a PCR-RFLP method for distinguishing four types of pratylenchus. A pair of common primers is designed based on the DNA sequence of the D2-D3 region of the 28S gene of nematode ribosome for PCR amplification by DNA extraction, then four types of restriction enzyme are utilized to carry out digestion and band spectrum identification so as to obviously distinguish P. coffeae, P. loosi, P. penetrans and P. vulnus. The PCR-RFLP method has stable results, simple operation, and wide application prospects in the fields of plant quarantine and plant protection.

Description

Technical field [0001] The present invention relates to the detection technology of Brachymeria nematodes, in particular to a PCR-RFLP method for distinguishing 4 species of Brachymeria nematodes. Background technique [0002] Brachyzoites ( Pratylenchus Filipjev, 1936) is an important type of migratory plant endoparasitic nematode, which can severely damage plant root tissue and cause root rot. At present, there are nearly 80 species of Nematodes of the genus Brachyma that have been found in the world. In 2007, my country included nematodes (non-Chinese species) of the genus Brachyura into the "List of Entry Phytosanitary Pests of the People's Republic of China". Coffea elegans P. coffeae Brachymys lucifera P. loosi Puncture P. penetrans Paratymida P. vulnus It is common in the world, and the frequency of interception at Chinese ports is very high. Although these 4 species of nematodes are not the subject of quarantine, accurate identification of them will help us to make...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/683C12Q1/6888C12Q2531/113C12Q2521/301C12Q2565/125
Inventor 顾建锋何洁刘乐乐
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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