Specific primer for quantitatively detecting yellow leaf curl virus, as well as application of specific primer

A technology for quantitative detection of yellow leaf curl virus, applied in the field of pathogen detection, can solve the problem of inability to distinguish between dead and live pathogens, and achieve the effect of accurate amplification

Inactive Publication Date: 2017-08-18
BEIJING PLANT PROTECTION STATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the common qPCR method can quantify the pathogenic bacteria, it cannot distinguish between the dead and the dead of the pathogenic bacteria (Zhang Zhenjia, Yu Jihua, Wang Xilin. Preliminary report on the control test of bacterial angular spot of cucumber[J]. Journal of Gansu Agricultural University, 1989, 4 :63-66.; Tong Tiezheng, Wu Shuxu, Li Dan, et al. Study on the disinfection characteristics of pathogenic bacteria based on PMA-quantitative PCR selective detection technology[J].Environmental Science,2011,32(4):1120-1126.)
Studies have shown that the DNA of pathogenic bacteria can remain intact within days or even weeks after death (Josephson K, Gerba C, Pepper I. Polymerase chain reaction detection of nonviable bacterial pathogens [J]. Applied and Environmental Microbiology, 1993, 59 (10 ):3513-3515.), so how to distinguish the life and death of pathogens has become a huge challenge for molecular detection methods

Method used

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  • Specific primer for quantitatively detecting yellow leaf curl virus, as well as application of specific primer
  • Specific primer for quantitatively detecting yellow leaf curl virus, as well as application of specific primer
  • Specific primer for quantitatively detecting yellow leaf curl virus, as well as application of specific primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] This example is used to illustrate the acquisition and verification of primer pairs for the quantitative detection of yellow leaf curl virus.

[0066] Cucumber leaves suffering from yellow leaf curl virus disease were obtained, put into a mortar, added liquid nitrogen to grind thoroughly, and DNA was extracted using a DP305 DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., adjusted The concentration of the DNA solution was 200 μg / μL.

[0067] Use the PA / PB degenerate primer pair and use the above-mentioned extracted DNA as a template to perform PCR amplification according to the following PCR amplification system and procedure:

[0068] PCR reaction system: (total volume 50μL)

[0069]

[0070] ddH 2 O to 50 μL, mix well, and centrifuge briefly.

[0071] PCR reaction program:

[0072] First, 94°C for 1min45s, and then (94°C for 30s, 45°C for 30s, 72°C for 30s) for 30 cycles.

[0073] in,

[0074] The PA primers and PB primers...

Embodiment 2

[0090] This example is used to illustrate the method for quantitative detection of yellow leaf curl virus provided by the present invention.

[0091] The cucumber leaves infected with yellow leaf curl virus are fumigated and sterilized with ozone according to the method of fumigation in the residue treatment machine (this treatment method can kill yellow leaf curl virus, and as the treatment time changes, the killed yellow leaf curl virus Leaf Curl Virus also can increase), handle respectively 0 minutes, 20 minutes, 30 minutes, 40 minutes and 50 minutes, after harvesting the blade after the treatment, each process is all processed as follows, to quantitatively detect Yellow Leaf Curl Virus content.

[0092] Put the cucumber leaves in a mortar, add liquid nitrogen and grind them thoroughly; take 50 mg of cucumber leaf powder and place them in EP tubes, and one of the tubes will be treated with PMA according to the following method for PMA-qPCR amplification:

[0093] Add 1 mL ...

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PUM

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Abstract

The invention belongs to the field of pathogen detection, and provides a primer pair for quantitatively detecting a yellow leaf curl virus, application of the primer pair to quantitative detection of the yellow leaf curl virus and a method for quantitatively detecting the yellow leaf curl virus. The primer pair for quantitatively detecting the yellow leaf curl virus can be used for efficiently and accurately amplifying a target fragment. The method for quantitatively detecting the yellow leaf curl virus by virtue of the primer pair can be used for accurately and rapidly identifying pathogenic bacteria and accurately quantifying a viable count.

Description

technical field [0001] The invention belongs to the field of pathogen detection, in particular to a specific primer for quantitative detection of yellow leaf curl virus and its application. Background technique [0002] Traditional detection methods of pathogenic bacteria, such as physiological and biochemical index identification methods, bacterial isolation and culture identification methods, etc., rely on phenotypes to identify pathogenic bacteria; these methods are time-consuming, have low sensitivity, and poor accuracy. [0003] Propidium azide bromide (PMA) is a fluorescent dye with high affinity to DNA, which can pass through the damaged cell membrane and covalently cross-link with DNA under the irradiation of visible light, so as to block the DNA in damaged cells. The role of DNA for PCR amplification. Some studies have reported that the PMA-qPCR method combined with the detection of live pathogen content, but only applied in water (Fujimoto J, Watanabe K. Quantitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2545/114C12Q2563/107
Inventor 李云龙何晓青王晓青金一王胤孔维文孙海刘婷婷胡彬郑建秋刘正雄李清波雷喜红陈娟王俊侠张保常张宁孙艳艳杨武群张超孙璐张宝杰李冬冬周长青吴继宗蔡乐饶玉燕
Owner BEIJING PLANT PROTECTION STATION
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