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, improve sensitivity, and sensitive primers

Active Publication Date: 2012-11-07
嘉兴卓十生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 sequence feature design of the D2D3 region. The nucleotide sequence of the primer is:

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

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

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

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

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

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

[0058] Specific detection of primers: BLAST (http: / / www.ncbi.nlm.nih.gov / blast) online comparison was performed first, and the primer pair NF3 / NR0 and root-knot nematode, reniform reniform nematode and other plant parasitic nematodes were found and the free-living rodent elegans are 100% similar. Root-knot nematode-specific primer MF0 and root-knot nematode M. M. enterolobii (JN00...

Embodiment 2

[0085] Example 2 Field application of the rapid detection method for root-knot nematode and reniform reniform nematode

[0086] For 57 cucumber rhizosphere soil samples collected from different regions in Guangdong, Guangxi, Yunnan, Hainan, and Inner Mongolia, the main plant parasitic nematode species were determined based on morphological observation combined with 28S D2D3 region molecular amplification and sequence comparison. 5g of fresh cucumber rhizosphere soil, using homemade extraction buffer Buffer H for rough DNA extraction method, Beijing Tiangen Biological Company's ordinary agarose gel DNA recovery kit purification, the purified product is directly subjected to nested multiplex PCR, and at the same time The sterilized soil without insects was used as negative control. 4 replicates per sample. The reaction conditions are the same as those described in "(3) Extraction method of total soil DNA". The test results are attached Figure 4 , with Figure 4 Among them...

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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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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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