Method capable of synchronously detecting RT-PCR detection primer group of two melon quarantine diseases, kit and application of method

A detection kit, RT-PCR technology, applied in the direction of microorganisms, recombinant DNA technology, microorganism-based methods, etc., to achieve the effects of reducing cost and time, ensuring sensitivity and specificity, and simplifying operations

Inactive Publication Date: 2017-07-14
INST OF PLANT PROTECTION GUANGXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved in the present invention is to establish a fast, specific and efficient multiplex PCR detection method on the basis of the RT-PCR of cucumber green mottle mosaic virus and

Method used

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  • Method capable of synchronously detecting RT-PCR detection primer group of two melon quarantine diseases, kit and application of method
  • Method capable of synchronously detecting RT-PCR detection primer group of two melon quarantine diseases, kit and application of method
  • Method capable of synchronously detecting RT-PCR detection primer group of two melon quarantine diseases, kit and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 : Design of primer sequences

[0038] According to the sequence design and optimization of cucumber green mottle mosaic virus (GQ277655.1) and cucurbit bacterial fruit spot disease (JQ901493.1) reported on NCBI, the specific pairing is shown in Table 1 below.

[0039] Table 1 Primer Sequence

[0040]

Embodiment 2

[0041] Example 2: Detection of known susceptible samples

[0042] Samples to be tested include: 1-Healthy gourd seeds; 2-Samples known to be infected with melon bacterial fruit spot; 3-Samples known to be infected with Cucumber green mottle mosaic virus; 4-Samples known to be simultaneously infected with Cucumber green mottle mosaic virus , Cucurbit bacterial fruit spot fungus samples.

[0043] Perform RT-PCR detection on the above samples respectively according to the following steps.

[0044] (1) Extract the total nucleic acid of the sample

[0045] Weigh 0.2g of the sample to be tested and place it in a mortar, add liquid nitrogen to grind, and quickly transfer the sample to a 1.5mL centrifuge tube containing 1mL TRNzol-A+ total nucleic acid extraction reagent after grinding, shake vigorously, add 200uL chloroform to vigorously Shake, centrifuge at 12000rpm at 4°C for 10min, take 400uL of supernatant to a new 1.5mL centrifuge tube, add 240uL of frozen isopropanol, mix b...

Embodiment 3

[0061] Example 3 : RT-PCR detection of known infected disease and unknown infected disease samples

[0062] Samples to be tested include: 1-Healthy gourd seeds; 2-Samples known to be infected with cucumber green mottle mosaic virus; 3-Samples known to be infected with cucurbit bacterial fruit spot; 1. Samples of bacterial fruit spot of melons; 5-8 are samples infected with unknown diseases.

[0063] With reference to the detection method in embodiment 1, each sample is carried out RT-PCR detection respectively, and detection result sees figure 2 .

[0064] Such as figure 2 Shown, can observe a 437bp nucleic acid band in the gourd seed sample (being sample 2) that only infects melon bacterial fruit spot bacterium, in the gourd seed sample that only infects cucumber green mottle mosaic virus (being sample 3 A nucleic acid band of 948bp can be observed in ), and two nucleic acid bands of 948bp and 437bp can be observed in the gourd seed sample (i.e. sample 4) infected with...

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Abstract

The invention belongs to the technical field of detection on melon quarantine diseases, especially relates to a method capable of synchronously and quickly detecting RT-PCR detection primer group of two melon quarantine diseases, i,e., cucumber green mottle mosaic virus (CGMMV) and acidovorax avenae subsp. citrulli (Aac), a kit and application of the method. According to the method provided by the invention, specific primers CGMMV-F/CGMMV-R, BFB-F/BFB-R are designed specifically for cucumber green mottle mosaic virus disease and acidovorax avenae subsp. Citrulli disease, and a RT-PCR detection technique is combined, synchronous detection and identification of the two diseases can be realized. The method provided by the invention is a specific, sensitive, convenient and cost-effective detection method which can synchronously and quickly detect CGMMV and Aac, can ensure the sensitivity and specificity of the detection, meanwhile, detection cost is reduced, time is saved.

Description

technical field [0001] The invention belongs to the technical field of detection of melon quarantine diseases, in particular to a simultaneous rapid detection of two kinds of quarantine diseases infecting melons, cucumber green mottle mosaic virus (CGMMV) and melon bacterial fruit spot disease (Aac) Methods. Background technique [0002] Cucumber green mottle mosaic mosaic virus (CGMMV), a short-stem Virus, whose genome is positive single-stranded RNA with a full length of 6.4Kb, belongs to the genus Tobamovirus of the family Tymoviridae. The virus mainly harms Cucurbitaceae crops and seriously threatens the production of watermelon, muskmelon, gourd, etc. It is mainly transmitted by seeds for long-distance transmission, and can also be transmitted by friction contact, soil diseased residues, grafting and pollination. The disease occurred on watermelons as early as 1968 in the Kanto area of ​​Japan, causing a loss of 900 million yen in the watermelon industry; in 1978, the...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143
Inventor 谢慧婷李战彪秦碧霞崔丽贤杨世安伍豪蔡健和
Owner INST OF PLANT PROTECTION GUANGXI ACADEMY OF AGRI SCI
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