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Detection method of symbiotic Wolbachia bacteria in mymanidae bodies

A detection method, the technology of tassels, applied in the fields of biochemical equipment and methods, determination/inspection of microorganisms, etc.

Inactive Publication Date: 2012-05-09
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

[0005] The defect of prior art is: the factors that affect PCR, such as the DNA template quality that extracts, are used for the primer and reagent of PCR, PCR reaction system, PCR reaction condition, the amplification efficiency of target gene in sample DNA etc. Affecting the final test result, the existing technology cannot ensure that the test result is not affected by the above factors in the experimental system, especially when a negative test result is obtained

Method used

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  • Detection method of symbiotic Wolbachia bacteria in mymanidae bodies
  • Detection method of symbiotic Wolbachia bacteria in mymanidae bodies
  • Detection method of symbiotic Wolbachia bacteria in mymanidae bodies

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Embodiment

[0033] 1. The source of the gnats used in this implementation example is the rice lice. T1 and T2 are collected from Zhejiang, which are samples cultivated at different temperatures. P5 is collected from the paddy field of the Philippine International Rice Research Institute Experimental Farm. The collected samples are preserved Store in -72°C refrigerator for later use.

[0034] 2. Genomic DNA extraction: Take out the cryopreserved wasp sample and put it on a clean bench to thaw. Take 10 samples of each type of wasp and put them into sterile 2 ml centrifuge tubes, add 1 mL of 70% ethanol, shake gently for 5 min, then rinse with sterile water for 3 times, repeat this step 3 times to remove the bacteria on the surface of the rice lice wasps.

[0035] Genomic DNA was extracted from samples of A. oryzae with DNeasy Blood & Tissue Kit (purchased from Qiagen Corp.). Put the wasp sample with surface bacteria removed into a new sterile 2 mL centrifuge tube, add 180 μL of ATL buffer...

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Abstract

The invention provided a detection method of a flora of symbiotic bacteria which is Wolbachia bacteria in mymanidae bodies. Specifically, the method comprises steps that: total DNA in a mymanidae sample is extracted; genetic segments of 16SrDNAV3 hypervariable regions of Wolbachia bacteria, total bacteria, and total bacteria except Wolbachia bacteria in the mymanidae sample are amplified through nested PCR; DGGE (denaturing gradient gel electrophoresis) analysis is carried out; and the Wolbachia bacteria in the mymanidae sample can be detected through DGGE spectrum analysis, and through the recovering, cloning and sequencing of the DGGE strap. The invention provides a relatively rigorous PCR-based technological method for detecting Wolbachia bacteria in mymanidae bodies. Also, with the method, varieties of Wolbachia bacteria in mymanidae bodies can be rapidly and accurately determined. Therefore, the method has an important promoting significance and a practical value on current detections and related researches of symbiotic Wolbachia bacteria in insect bodies.

Description

technical field [0001] The invention relates to the molecular detection of symbiotic bacteria in insects, in particular to the symbiotic bacteria Wolbachia ( Wolbachia ) Bacteria detection method. Background technique [0002] The wasp belongs to the family Mymanidae of the order Hymenoptera, and is an important parasitic wasp in the egg stage of the main rice pest planthopper. The large number of syringus wasps and high efficiency have a significant inhibitory effect on the occurrence of SBPH and white-backed planthopper in the early rice field and the brown planthopper in the late rice field. Therefore, the research on the wasp is of great significance in the biological control of rice pests and diseases. Existing studies have shown that there are a large number of symbiotic microorganisms in parasitic wasps, among which Wolbachia ( Wolbachia ) is the most widely distributed bacteria, which can induce thelytoky and cytoplasmic incompatibility of parasitoid wasps, cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 吕仲贤汤江武徐红星王新郑许松刘淑平杨亚军
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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