One-step multi-PCR detection method for simultaneously detecting four important citrus pathogenies

A detection method, citrus technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of one-step multiple PCR system for grafting transmission diseases that have not yet been seen, achieve efficient and rapid detection, avoid repeated detection, The effect of ensuring safe production

Inactive Publication Date: 2012-07-18
CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0005] At present, the single RT-PCR molecular detection technology for CTV, CEVd, and CTLV has been established, and the PCR technology for Huanglongbing has been established, but there are no reports on the one-step multiplex PCR system for the four important graft-transmitted diseases.

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  • One-step multi-PCR detection method for simultaneously detecting four important citrus pathogenies
  • One-step multi-PCR detection method for simultaneously detecting four important citrus pathogenies
  • One-step multi-PCR detection method for simultaneously detecting four important citrus pathogenies

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Embodiment Construction

[0047] The present invention simultaneously detects 4 kinds of important grafting-transmitted pathogens of citrus in one-step multiple PCR detection method as follows:

[0048] 1. Nucleic acid extraction: (1) From citrus samples picked in Jiangxi, Yunnan, Hunan, Zhejiang, Chongqing, Guangdong, Guangxi and other places, 13 samples were randomly selected, and 10-15 mg of skin and veins were taken from each sample in sterile eppendorf tube, ground with liquid nitrogen;

[0049] (2) Add 60 μL TES buffer and 60 μL saturated phenol: chloroform: isoamyl alcohol (25:24:1) in sequence, mix well,

[0050] (3) 70°C water bath for 8 minutes, centrifuged at 12000rpm for 5 minutes,

[0051] (4) Pipette 40 μL of supernatant into a Sephadex G-50-80 chromatography column, centrifuge at 5000 rpm for 4 min, collect the eluate, and store at -20°C.

[0052] The above-mentioned chromatographic column is prepared as follows: add a small amount of TNE-balanced glass beads to the bottom of a 0.5mL E...

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Abstract

The invention discloses a one-step multi-PCR detection method for simultaneously detecting four important citrus pathogenies. According to the method, primers for the four important pathogenies including Citrus tristeza virus, Citrus exocortis viroid, Citrus tatter-leaf virus and Liberobacter asiaticum spreading through grafting are preferably selected, and primer concentration, PCR amplificationcondition and the like are optimized, therefore, the four important citrus pathogenies spreading through grafting can be simultaneously detected. The detection method provided by the invention is quick and accurate, has the advantages of good detection stability, high specificity and high consistency, is suitable for large-scale generalization, and can be used for guiding the introduction of varieties and generalization of detoxifying treatment of seedlings to ensure the safe production of citrus; in addition, by adopting the detection method, repeated detection in the conventional PCR is avoided.

Description

technical field [0001] The invention belongs to the field of agricultural molecular biology, and specifically relates to a one-step multiplex PCR detection method for simultaneously detecting four important graft-transmitted pathogens of citrus. Background technique [0002] There are currently 5 kinds of graft-transmitted diseases identified in my country, including Huanglongbing, decaying disease, broken leaf disease, split skin disease and Wenzhou citrus shrunken disease. Due to the application of virus-free seedlings, the occurrence and distribution of Wenzhou satsuma shrunken disease in the field is rare. Citrus decline disease is an important disease caused by Citrus tristeza virus (CTV). The safety of grapefruit, sweet orange and other varieties sensitive to CTV stem pit strains. Citrus exocortis viroid is a worldwide important citrus disease caused by Citrus exocortis viroid (CEVd), and it is an important graft-transmissible viroid that limits the production of cit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
Inventor 刘金香周常勇李中安唐科志王雪峰
Owner CITRUS RES INST OF CHINESE ACAD OF AGRI SCI
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