LAMP (loop-mediated isothermal amplification) rapid detection method for squama corm nematode

A detection method, stem nematode technology, which is applied in the field of molecular biology detection of plant diseases, can solve the problem of high equipment requirements, and achieve the effects of high sensitivity, simple result determination, and strong specificity

Pending Publication Date: 2022-05-24
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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AI Technical Summary

Problems solved by technology

However, the conventional specific primer PCR method has high requirements for equipment: PCR instrument, gel electrophoresis instrument and other equipment are required

Method used

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  • LAMP (loop-mediated isothermal amplification) rapid detection method for squama corm nematode
  • LAMP (loop-mediated isothermal amplification) rapid detection method for squama corm nematode
  • LAMP (loop-mediated isothermal amplification) rapid detection method for squama corm nematode

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Nematode DNA extraction

[0057] The nematode DNA was extracted using the DNeasy Blood&Tissue Kit (QIAGEN) kit, and the extraction method was carried out according to the instruction manual of the kit.

[0058] 2. LAMP primer design

[0059] LAMP primer design is more complicated than conventional PCR methods. Generally, the reaction includes at least 4 specific primers: outer primers (F3, B3) and inner primers (FIP, BIP), which are designed for 6 regions on the gene, respectively located in 3 The F3c segment at the 'end, the F2c segment, the F1c segment and the B1 segment at the 5' end, the B2 segment, the B3 segment. Sometimes, the LAMP reaction can be promoted by adding two loop primers (F-Loop primer, B-Loop primer), which can increase the reaction speed by 1 / 2 to 1 / 3.

[0060] According to the sequence of D. dipsaci and its related species published by NCBI, a lot of research and analysis have been carried out, including the comparison within the genus of nema...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) rapid detection method of squama corm nematode, and belongs to the field of molecular biological detection of plant diseases. Comprising the following steps: 1) taking DNA of a sample to be detected as a template, and carrying out LAMP amplification by utilizing an LAMP primer group of the squama corm nematode; 2) judging whether the sample to be detected contains the squama corm nematode or not according to whether specific amplification occurs or not; wherein the LAMP primer group comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a pair of loop primers LF / LB, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO.1-SEQ ID NO.6. The LAMP primer group comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a pair of loop primers LF / LB. The method is rapid and convenient; the specificity is high; the sensitivity is high; the steps are simple; result determination is simple; meanwhile, the method is safe to human and environment, toxic reagents such as EB do not need to be used in the detection process, and the detection requirement for rapidly and accurately identifying the squamous corm stem nematode in a first-line laboratory of inspection and quarantine can be met.

Description

technical field [0001] The invention belongs to the field of molecular biology detection of plant diseases, in particular to a LAMP rapid detection method of D. dipsaci. Background technique [0002] Ditylenchus dipsaci belongs to the phylum nematodes, Ditylenchus, Granulaceae, and the genus Ditylenchus. It can cause a variety of plant destructive diseases. It is a world-recognized dangerous nematode and is listed as a quarantine pest in my country. directory. The host range of D. bulbi is extremely wide, and it can parasitize about 500 kinds of plants in about 40 families, including sweet potatoes, potatoes, oats, beets, onions, narcissus, hyacinths, tulips, gladiolus, and red tops. [0003] Studies have shown that when there are 10 nematodes per 500g of soil, it can seriously damage plants such as onions, beets, and carrots, and the loss can reach 60% to 80% in severe infection. In Italy, the nematode infests onions, with a mortality rate of 60% at the seedling stage befo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2527/101C12Q2531/119C12Q2563/107Y02A50/30
Inventor 俞禄珍宋绍禕李帅于翠王一椒李丽
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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