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Molecular detection primer of acidovorax avenae subsp. Citrulli and application thereof

A technology for the detection of fruit spot bacteria and molecules, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of high false positive rate, low sensitivity, and poor specificity, and achieve high sensitivity , reliable results, and easy-to-operate results

Inactive Publication Date: 2010-12-01
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a watermelon bacterial fruit spot pathogen molecular detection primer and its application, aiming at the long period required for the biological detection method of watermelon bacterial fruit spot pathogen in the prior art, the current watermelon bacterial fruit spot pathogen molecular The detection method has poor specificity, high false positive rate, and low sensitivity, and provides a specific molecular detection primer for bacterial fruit spot of watermelon

Method used

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  • Molecular detection primer of acidovorax avenae subsp. Citrulli and application thereof
  • Molecular detection primer of acidovorax avenae subsp. Citrulli and application thereof
  • Molecular detection primer of acidovorax avenae subsp. Citrulli and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Primer is specific to the amplification of watermelon bacterial fruit spot bacterium

[0037] 1. Specific detection of watermelon bacterial fruit spot

[0038] The DNA extraction method of each strain was carried out according to the above-mentioned SDS-CTAB method, and the PCR reaction system was 25 μL, including 2.5 μL 10×PCR reaction buffer, 2.0 mmol / L Mg 2+ , 150 μmol / L dNTPs, 1.25U Taq DNA polymerase, 0.2 μmol / L each of primers AIT1F and AIT2R and 10 ng template DNA, d.d.H 2 O was added to 25 μL, and the PCR reaction conditions were: 94 °C pre-denaturation for 4 min, 94 °C denaturation for 35 s, 65 °C annealing for 35 s, 30 cycles, and 72 °C extension for 5 min.

[0039] 2. Test results

[0040] Specificity of detection: In addition to the 462 bp product that can be amplified specifically for bacterial fruit spot of watermelon, other 6 strains of acidovorous bacteria, Ralstonia solanacearum, Pseudomonas rhodostripe, and Pseudomonas syringae wer...

Embodiment 2

[0041] Embodiment 2: Sensitivity detection of primers to bacterial fruit spot of watermelon

[0042] 1. Detection of bacterial concentration dilution and lysis: the culture solution of watermelon bacterial fruit spot bacteria was diluted to 10 1 、10 2 、10 2 、10 4 、10 5 、10 6 、10 7 、10 8 CFU / mL, after lysing with boiling water, directly draw the supernatant for PCR detection.

[0043] 2. Sensitive detection of watermelon bacterial fruit spot

[0044] PCR reaction system 25 μL, including 2.5 μL 10×PCR reaction buffer, 2.0 mmol / L Mg 2+, 150 μmol / L dNTPs, 1.25U Taq DNA polymerase, 0.2 μmol / L each of primers AIT1F and AIT2R and 1 μL bacterial lysate as template DNA, d.d.H 2 O was added to 25 μL, and the PCR reaction conditions were: 94 °C pre-denaturation for 4 min, 94 °C denaturation for 35 s, 65 °C annealing for 35 s, 30 cycles, and 72 °C extension for 5 min.

[0045] 3. Test results: 10 5 Bands can be seen at the CFU / mL concentration, that is, in a 25 μL react...

Embodiment 3

[0046] Embodiment 3: detection of watermelon bacterial fruit spot pathogen in diseased plant tissue

[0047] 1. Sample collection: Plant tissue samples of diseased watermelon were collected from Minqing Vegetable Base in Fujian Province.

[0048] 2. DNA extraction and detection

[0049] The diseased plant tissue adopts DNA rapid extraction method to obtain the NDA of watermelon fruit spot bacteria in the diseased tissue. The specific method is to take 200 mg of diseased leaf or fruit tissue, add 2 mL of 0.5 mol / L NaOH to grind; take 5-l to grind The solution was added to 495 - l Tris-HCl (pH 8.0) buffer solution with a concentration of 0.1 mol / L, centrifuged at 12,000 rpm for 3 min, and the supernatant was taken for PCR reaction. Carry out PCR amplification according to the above method, PCR reaction system 25 μL, including 2.5 μL 10×PCR reaction buffer, 2.0 mmol / L Mg 2+ , 150 μmol / L dNTPs, 1.25U Taq DNA polymerase, 0.2 μmol / L each of primers AIT1F and AIT2R and 10 ng te...

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Abstract

The invention provides a molecular detection primer of acidovorax avenae subsp. Citrulli and application thereof. The special primer comprises an upstream primer AIT1F: 5'-GCTGGATCACCTCCTTTCTG-3' and a downstream primer AIT2R: 5'-TGACGCAATCAAATTTTTGTCA-3'. Through PCG (Polymerase Chain Reation) amplification and agarose gel electrophoresis, a special amplification product with a fragment length of 462bp can be amplified in pure DNA of the acidovorax avenae subsp. Citrulli, a bacteria carrying disease plant tissue and a seed to rapidly detect the acidovorax avenae subsp. Citrulli. The special detection primer and the application thereof can be used for rapidly, sensitively and specially detecting the acidovorax avenae subsp. Citrulli in bacteria carrying watermelon seeds and plant seeds infected the acidovorax avenae subsp. Citrulli in production practice, can also be used for early diagnosis of field diseases and monitoring and identification of germs and provide a reliable technology and theoretical basis to control diseases caused by the acidovorax avenae subsp. Citrulli.

Description

technical field [0001] The invention belongs to the field of crop disease detection, identification and prevention technology, and more specifically relates to a molecular detection primer for watermelon bacterial fruit spot disease and its application. Background technique [0002] Bacterial fruit spot of watermelon is caused by Acidovorax subsp. Acidovorax avenae subsp. citrulli , referred to as A.a.c) is a disease caused by infestation, which can seriously damage the leaves and fruits of watermelon, resulting in a large reduction in yield. It was first discovered in Florida, USA by Crall and Schenck in 1969. They did not identify the pathogenic bacteria, but they only described the symptoms of the disease. In 1988, Wall and Santos reported the occurrence of watermelon bacterial fruit spot in Guam, and identified the pathogen for the first time as Pseudomonas subsp. Pseudomonas pseudoalcaligenes subsp. citrulli ). In 1992, according to the research results of rRNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 邱思鑫陈新凯
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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