Primers and method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil

A root-knot nematode and reniform nematode technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problem of not being able to directly detect root-knot nematode and reniform reniform nematode at the same time , to achieve high practical application value, shorten sample detection time, and sensitive primers

Active Publication Date: 2013-12-25
嘉兴卓十生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a group of primers that can directly and simultaneously detect root-knot nematode and reniform nematode in soil simultaneously for the deficiency that root-knot nematode and reniform reniform nematode can not be directly and simultaneously detected from soil in the prior art

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  • Primers and method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil
  • Primers and method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil
  • Primers and method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil

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Experimental program
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Effect test

Embodiment 1

[0050] (1) Primer design and synthesis

[0051] 根据植物线虫大亚基核糖体DNA(GenBank 登录号:JF461074-JF461078,AF435797-AF435800,EU364889,AF435803,AF435794,GQ130139,DQ145641,DQ328685,HQ420904,HQ420905,AF435793,AF435801,DQ328713,GU120091,HM131853- HM131884) The D2D3 region sequence feature design. The nucleotide sequences of the primers are:

[0052] NF3: 5'-GTGAGGGAAAGTTGCAAAGCACT-3';

[0053] NR0: 5'-GTTCACCATCTTTCGGGTCTCAC-3';

[0054] MF0: 5'-GGGGATGTTTAGGCAGATTTG-3';

[0055] RF4: 5'-GGTCTGGCTCCCATGTTTTCCA-3';

[0056] D2A: 5'-ACAAGTACCGTGAGGGAAAGTTG-3';

[0057] D3B: 5'-TGCGAAGGAACCAGCTACTA-3'.

[0058] Specificity detection of primers: BLAST (http: / / www.ncbi.nlm.nih.gov / blast) online comparison was performed first, and the primer pair NF3 / NR0 and plant parasitic nematodes such as root-knot nematode and reniform nematode were found. and the free-living rodent nematodes are 100% similar. Root-knot nematode specific primer MF0 and root-knot nematode nematode root-knot nematode M. ...

Embodiment 2

[0085] Example 2 Field application of rapid detection methods for root knot nematodes and reniform nematodes

[0086] For 57 cucumber rhizosphere soil samples collected from different regions in Guangdong, Guangxi, Yunnan, Hainan, Inner Mongolia and other provinces, the main plant parasitic nematode species were firstly determined based on morphological observation combined with molecular amplification and sequence alignment in the 28S D2D3 region, and then the 5 g of fresh cucumber rhizosphere soil was used for crude DNA extraction with the self-made extraction buffer Buffer H, purified by the ordinary agarose gel DNA recovery kit of Beijing Tiangen Biological Company, and the purified product was directly subjected to nested multiplex PCR. Sterilized soil without worms was used as a negative control. 4 replicates per sample. The reaction conditions were the same as those described in the aforementioned "(3) Extraction method of total soil DNA". The test results are attac...

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Abstract

The invention discloses primers and a method for directly and simultaneously detecting Meloidogyne and Rotylenchulus reniformis in soil and belongs to the technical field of molecular biology detection. Nucleotide sequences of the primers for directly and simultaneously detecting the Meloidogyne and the Rotylenchulus reniformis in the soil are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively. The invention also discloses the method for directly and simultaneously detecting the Meloidogyne and the Rotylenchulus reniformis in the soil. The method comprises the following steps of: directly extracting deoxyribonucleic acid (DNA) from soil, purifying, performing first-round polymerase chain reaction (PCR) by using the universal primers of nematodes, namely D2A and D3B by taking the purified DNA as a template, performing second-round PCR by using the primers which are shown as SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 by taking a product as a template, and judging whether the Meloidogyne and the Rotylenchulus reniformis are contained in a sample through the magnitude of fragments of a PCR product. The primers are sensitive and high in specificity; by the method for directly and simultaneously detecting the Meloidogyne and the Rotylenchulus reniformis in the soil, the detection time can be greatly saved, and results are accurate and reliable; and the primers have high practical application value in the aspect of the rapid detection of the Meloidogyne and the Rotylenchulus reniformis.

Description

technical field [0001] The invention relates to the technical field of detecting plant harmful nematodes by means of molecular biology methods, in particular to primers and a method for directly and simultaneously detecting root-knot nematodes and reniform reniform nematodes in soil. Background technique [0002] root-knot nematodes ( 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. reniform reniform nematode ( Rotylenchulus reniformis ), as a model species of the genus Reniformis, it is seriously harmful to agricultural production, mainly occurs in tropical and subtropical regions, and has a wide range of hosts. Infection and parasitism on vegetable crops and a variety of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
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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