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Method for detecting single root knot of enterolobium cyclocarpum meloidogyne and application thereof

A root-knot nematode and detection method technology, applied in the biological field, can solve the problem of accurate identification of template DNA from nematodes and root-knot nematodes, achieve high practical application value, reduce detection time, and improve sensitivity

Inactive Publication Date: 2013-01-30
嘉兴卓十生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, conventional identification methods are difficult to accurately identify root-knot nematode, because the perineal pattern of root-knot nematode is very similar to that of root-knot nematode incognita
Molecular identification can basically satisfy the detection and identification of root-knot nematode, but the template DNA is all from the nematode

Method used

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  • Method for detecting single root knot of enterolobium cyclocarpum meloidogyne and application thereof
  • Method for detecting single root knot of enterolobium cyclocarpum meloidogyne and application thereof
  • Method for detecting single root knot of enterolobium cyclocarpum meloidogyne and application thereof

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Experimental program
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Effect test

Embodiment 1

[0038] (1) Obtaining single knot DNA

[0039] Sow tomato (Xiahong No. 1), cucumber (Xinwanji) and water spinach 25 days later and transplant the seedlings to flower pots with sterilized soil with a diameter of 15 cm, 1 plant per pot. One week after transplanting, each plant was inoculated with 200 2nd instar larvae (J2) of Meloidogyne elegans, Meloidogyne incognita, Meloidogyne javanica and Root-knot nematode peanut, while keeping healthy plants without inoculation as a control , placed in a light culture room for 14 hours of light and 10 hours of darkness for constant temperature cultivation at 25°C. The morphology of nematodes was observed by staining at 5d, 10d, 15d, 20d, 25d, and 30d after inoculation, respectively. At the same time, healthy root tissue DNA and single root junction DNA were extracted, and 5 replicates were taken for each treatment, and 1 blank control was set.

[0040] Root tissue staining and nematode morphology observation at different stages, the step...

Embodiment 2

[0050] (2) Synthetic primers

[0051] 根据植物线虫大亚基核糖体DNA(基因登录号为:AF435803、AF435794、AY446970、JN005857、FN429017、GQ375158、EU364890、EU570214、AF435793、AF435802、FJ485651、DQ328713、HQ688681、EU130893和EU36859)的D2D3区设计线虫 通用引物MF和MR;根据根结线虫mtDNA COII和lrRNA基因(基因登录号为:FJ159631、AY635612、GQ266686、GQ266685、AY446970、GQ870255、AY757882、GQ865513、HM161680、AY635609、AY757909、AY942848、AY942851和EU364883)间 The specific primers FMe and Rme of Meloidogyne elegans were designed based on regional interspecific differences.

[0052] The nucleotide sequence of the primer is as follows:

[0053] MF 5'-GGGGATGTTTGAGGCAGATTTGT-3';

[0054] MR 5'-AACCGCTTCGGACTTCCACCAG-3';

[0055] FMe 5'-CATTCATTTATACCAATTTTAGTTGAGG-3';

[0056] RMe 5'-CAATTATTGAATATTTTTTTCCCAAACGA-3'.

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Abstract

The invention discloses a method for detecting a single knot of enterolobium cyclocarpum meloidogyne and application thereof. In the invention, universal primers MF (Forward Primer) and MR (Reverse Primer) for amplifying meloidogyne and specific primers FMe (Forward Primer) and RMe (Reverse Primer) for amplifying the enterolobium cyclocarpum meloidogyne are designed; a single root knot DNA containing the meloidogyne is taken as a template for PCR (Polymerase Chain Reaction) amplification reaction; a PCR amplification reaction product is subjected to lipolysaccharide electrophoresis detection, and a detection result is obtained according to the existence / absence and the size of straps; and the sequences of the primers are respectively shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 in a table. The one-step dual PCR detection method disclosed by the invention has the advantages of increased molecular detection speed, increased sensitivity and accuracy, and has a practical application value in the aspect of detecting the enterolobium cyclocarpum meloidogyne.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a detection method and application of root-knot nematode Elephant ear. Background technique [0002] Root-knot nematode (Meloidogyne) is an obligate endoparasite of plant roots, distributed throughout most parts of the world. Because of its strong adaptability, diverse transmission routes, and ability to infect a variety of plants, it has become the main pathogen that threatens agricultural production worldwide, causing great economic losses to agricultural producers. There are many kinds of root-knot nematodes in my country and they are widely distributed, except for the common root-knot nematodes (M. incognita), root-knot nematodes Java (M. javanica), root-knot nematodes northern (M. arenaria) 4 major root-knot nematodes, surveys in recent years have shown that root-knot nematode M. enterolobii is widely distributed in southern my country and is seriously harmfu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 廖金铃胡茂秀卓侃
Owner 嘉兴卓十生物科技有限公司
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