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DPO-PCR primer and method for detecting coix lacryma-jobi leaf spot bacteria

A technology of DPO-PCR and Coix leaf spot bacteria, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of poor specificity, long cycle time, and low sensitivity of detection methods, and achieve specificity Strong, reliable results and high sensitivity results

Pending Publication Date: 2022-05-10
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a highly specific DPO-PCR detection method for Coix leaf spot fungus aiming at the problems of long period required, poor detection method specificity and low sensitivity of the biological detection method of Coix leaf spot fungus in the prior art Primers, and establish a DPO-PCR detection method based on agarose gel electrophoresis

Method used

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  • DPO-PCR primer and method for detecting coix lacryma-jobi leaf spot bacteria
  • DPO-PCR primer and method for detecting coix lacryma-jobi leaf spot bacteria
  • DPO-PCR primer and method for detecting coix lacryma-jobi leaf spot bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Specific amplification of DPO-PCR primers to Coix leaf spot bacteria

[0041] 1. DPO-PCR Specific Detection of Coix Leaf Spot

[0042] ①DPO-PCR reaction system 20 μL: containing 0.2 μM each of CcDPO-F and CcDPO-R, 2× Taq PCR MasterMix 10 μL, 25 ng DNA template, make up the deficiency with sterile double distilled water. The DPO-PCR reaction program was: 94°C pre-denaturation for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 20 s, 28 cycles; 72°C for 5 min; 12°C forever.

[0043] ②Take 5 μL of the amplified product and use 2% agarose gel electrophoresis to detect it. If a single band with a size of about 200 bp appears, it is judged as positive, and if no amplified band appears, it is judged as negative.

[0044] 2. Test results

[0045] Specificity of detection: In addition to the single band of about 200 bp in the DPO-PCR product of coix leaf spot bacteria after agarose gel electrophoresis, three pathogenic strains of the same genus and nine different...

Embodiment 2

[0046] Example 2: Sensitivity detection of DPO-PCR primers to Coix leaf spot fungus

[0047] 1. DPO-PCR Sensitive Detection of Coix Leaf Spot

[0048] The extracted Coix leaf spot DNA was diluted into 50 ng / µL, 10 ng / µL, 1 ng / µL, 100 pg / µL, 10 pg / µL, and 1 pg / µL with a total of 6 different Concentration gradient.

[0049] ①DPO-PCR reaction system 20 μL: containing 0.2 μM each of CcDPO-F and CcDPO-R, 2× Taq 10 μL of PCR MasterMix, 1 μL of DNA template, make up the deficiency with sterile double distilled water. The DPO-PCR reaction program was: 94°C pre-denaturation for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 20 s, 28 cycles; 72°C for 5 min; 12°C forever.

[0050] ②Take 5 μL of the amplified product and use 2% agarose gel electrophoresis to detect it. If a single band with a size of about 200 bp is judged as positive, if no amplified band appears, it is judged as negative.

[0051] 2. Test results: DPO-PCR sensitivity detection of Coix leaf spot fungus, a single b...

Embodiment 3

[0052] Example 3: Detection of the annealing temperature sensitivity of DPO-PCR primers to Coix leaf spot fungus

[0053] 1. DPO-PCR Annealing Temperature Sensitivity Detection of Coix Leaf Spot

[0054] ①DPO-PCR reaction system 20 μL: containing 0.2 μM each of CcDPO-F and CcDPO-R, 2× Taq PCRMaster Mix 10 μL, 25 ng DNA template, make up the deficiency with sterile double distilled water. The DPO-PCR reaction program was: 94°C pre-denaturation for 5 min; 94°C for 30 s, 45-65°C for 30 s, 72°C for 20 s, 28 cycles; 72°C for 5 min; 12°C forever, where the annealing temperature was set at 45°C, 8 different temperatures of 47°C, 49°C, 55°C, 57°C, 60°C, 63°C, 65°C.

[0055] ②Take 5 μL of the amplified product and use 2% agarose gel electrophoresis to detect it. If a single band with a size of about 200 bp appears, it is judged as positive, and if no amplified band appears, it is judged as negative.

[0056] 2. Detection results: In the DPO-PCR annealing temperature sensitivity ...

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Abstract

The invention discloses a DPO-PCR primer and a method for detecting coix lacryma-jobi leaf spot bacteria, provides a highly specific DPO-PCR detection primer for the coix lacryma-jobi leaf spot bacteria and establishes DPO-PCR detection based on agarose gel electrophoresis judgment. According to the invention, a pair of dual-start oligonucleotide (DPO) PCR detection primers is designed to detect the leaves and seeds of coix lacryma-jobi with bacteria, and whether the pathogenic bacteria are the leaf spot disease of coix lacryma-jobi is determined according to the existence of amplification products and the size of fragments. Aiming at the problems of long required period, poor detection method specificity and low sensitivity of a biological detection method of coix lacryma-jobi leaf spot bacteria in the prior art, compared with a traditional detection method, the DPO-PCR detection method provided by the invention is simple and convenient to operate and accurate in result.

Description

technical field [0001] The invention belongs to the technical field of plant protection, and in particular relates to a DPO-PCR primer and a method for detecting Coix leaf spot fungus. Background technique [0002] Job's tears ( Coix lacryma-jobi L), also known as Coix, Coix, Coix, Coix, etc., belongs to Gramineae Coix genus Coix , is a traditional medicine and food in my country. It has the effects of diuresis and dampness, clearing lung heat, and strengthening the spleen and stomach. Job's tears are planted in Fujian, Guizhou, Yunnan, Guangxi and other places in my country. Due to the rich nutritional value and medicinal efficacy of coix seed, the demand for coix coix has risen sharply, and the planting scale has continued to expand. [0003] In recent years, the disease of Coix coix leaf spot in Fujian has been serious. It causes water-soaked light yellow spots in the early stage of the disease, and in severe cases, it causes yellow-brown lesions until death. The dis...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2565/125Y02A50/30
Inventor 谢世勇王荣波季洁黄建成
Owner INST OF PLANT PROTECTION FAAS
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