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Polymerase chain reaction (PCR) authentication primer and method for distinguishing bradysia odoriphaga larva from bradysia difformis larva

One larvae, one-pair technology, applied in the field of agricultural biology

Inactive Publication Date: 2014-08-06
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But utilize PCR-RFLP technology to construct the method for distinguishing leek maggot and bacterial maggot and have not yet been reported

Method used

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  • Polymerase chain reaction (PCR) authentication primer and method for distinguishing bradysia odoriphaga larva from bradysia difformis larva
  • Polymerase chain reaction (PCR) authentication primer and method for distinguishing bradysia odoriphaga larva from bradysia difformis larva
  • Polymerase chain reaction (PCR) authentication primer and method for distinguishing bradysia odoriphaga larva from bradysia difformis larva

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Extraction of genomic DNA of chive maggots and bacterial maggots

[0037] Place the single-headed chive maggots and bacterial maggots in 0.2ml centrifuge tubes containing 60 μl of alkaline lysate, respectively: 50 mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA (ethylenediaminetetraacetic acid) and 1% SDS (sodium dodecyl sulfate) are fully ground and homogenized with a sealed gun head, placed in a water bath at 65°C for 15 minutes, and then placed in a water bath at 95°C for 10 minutes to obtain Chive maggot and bacterial maggot genome DNA solution.

[0038] (2) PCR amplification of COI genes of chive maggots and bacterial maggots

[0039] Carrying out PCR amplification with the chive maggot genome DNA solution and the bacteria maggot genome DNA solution as templates respectively to obtain PCR amplification products;

[0040] The PCR amplification system is:

[0041] Chive maggot genomic DNA solution: 3μl; 20μM primer: 0.5μl; 5U / μl Taq enzyme: 0.5...

Embodiment 2

[0049] A method for distinguishing leek maggots and bacterial maggots, the steps are as follows:

[0050] (1) Place the individual leek maggots and bacterial maggots collected in Jinan City, Shandong Province into 0.2ml centrifuge tubes containing 60μl of alkali lysis solution, the alkali lysis solution is: 50mmol L -1 Tris-HCl (pH8.0), 20mmol·L -1 NaCl, 1mmol L -1 EDTA, 1% SDS, fully grind the homogenate with a sealed pipette tip, put it in a water bath at 65°C for 15 minutes, and then put it in a water bath at 95°C for 10 minutes to obtain a genomic DNA solution;

[0051] (2) Using the genomic DNA prepared in step (1) as a template, performing PCR amplification on the mitochondrial COI gene in the genomic DNA to obtain a PCR amplification product;

[0052] The PCR amplification system is:

[0053] Genomic DNA solution 2μl, 20μM primer 0.5μl, 5U / μl Taq enzyme 0.25μl, 10×Taq Buffer 2.5μl, 10mM dNTP 0.5μl, ddH 2 0 to 25 μl;

[0054] The primer sequences are as follows:

...

Embodiment 3

[0063] The method for identifying leek maggots and bacterial maggots as described in Example 2, the difference is that the leek maggots and bacterial maggots were collected in Jinan City, Shandong Province in 2011.

[0064] The results show that when there are two bands of about 200bp and 400bp on the imaging film, it is a leek maggot; when there is a band with a fragment length of about 600bp on the imaging film, it is a maggot, and the results are as follows figure 1 As shown, and in accordance with (Shi Baocai, Lu Hong, Gong Yajun, etc., 2010. Identification and control of Chive tardigrades. Chinese Vegetables, 11:21-22.; Zhang Hongrui, Zhang Xiaoyun, Shen Dengrong, etc., 2008. Edible mushrooms Biological characteristics of the eye fungus Bradysia difformis. Chinese Edible Fungi, 27 (6): 54-56.) The detection results were consistent with the method recorded.

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Abstract

The invention relates to a polymerase chain reaction (PCR) authentication primer and method for distinguishing bradysia odoriphaga larva from bradysia difformis larva. The method comprises the following steps of: (1) extracting genome DNAs of the bradysia odoriphaga larva and the bradysia difformis larva; (2) carrying out PCR amplification on a mitochondria COI gene with the genome DNA of the bradysia odoriphaga larva and the bradysia difformis larva as a template; (3) carrying out enzyme digestion on a PCR product prepared in step (2) by using restriction enzyme EcoRV to obtain an enzyme-digested product; and (4) carrying out agarose gel electrophoresis analysis on the enzyme-digested product prepared in step (3). The restriction enzyme refers to a frequently-used restriction enzyme. By using the method, a simple, convenient and stable enzyme digestion sign is provided for screening the bradysia odoriphaga larva and the bradysia difformis larva, an technique for authenticating the bradysia odoriphaga larva and the bradysia difformis larva is explored and established, and a foundation is laid for the future bradysia odoriphaga larva and bradysia difformis larva authentication, population dynamic monitoring and comprehensive prevention and control.

Description

technical field [0001] The present invention relates to a PCR identification primer and an identification method for distinguishing tardigrade maggots and Heterophthalmia larvae, and in particular to a PCR-RFLP-based primer and identification method for identifying chive maggots and fungus maggots, belonging to agricultural biology. technology field. Background technique [0002] The larvae of Bradysia odoriphaga, commonly known as chive maggots, are the main pests of chives. The larvae harm leek leaf sheaths, young buds and bulbs, causing bulbs to rot and leaves to die. The mild ones cause lodging and yellowing, and the severe ones lack seedlings and break ridges, seriously affecting the yield and quality of leeks. [0003] The larvae of Bradysia difformis, commonly known as maggots, belong to the same genus Bradysia difformis as Bradysia difformis. The traditional method of distinguishing chive maggots from fungus maggots is based on their morphological characteristics (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 褚栋于毅国栋陶云荔
Owner QINGDAO AGRI UNIV
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