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Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare

A technology for anthracnose bacteria and detection primers is applied in the field of crop disease detection, identification and prevention, which can solve the problems of cumbersome procedures, disease decay and deterioration, and low accuracy, and achieves strong specificity and accuracy, strong specificity, and high sensitivity. Effect

Active Publication Date: 2015-11-18
INST OF PLANT PROTECTION FAAS
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] The purpose of the present invention is to detect and identify the guava anthracnose bacteria in the prior art, mainly based on morphological characteristics, the method is time-consuming, the procedure is loaded down with trivial details, experience is strong, accuracy is low, it is difficult to accomplish the timely monitoring and identification of disease occurrence. To control the spread and prevalence of pathogenic bacteria and the problems of rot and deterioration caused by postharvest diseases, a kind of PCR detection primer and detection method specific for guava anthracnose bacteria is provided, and the PCR detection primer and detection method described in the present invention are used to detect guava anthrax High accuracy, strong specificity, high sensitivity, easy operation, short detection time and reliable results

Method used

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  • Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare
  • Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare
  • Guava colletotrichum orbiculare specificity PCR detecting primer and detecting method of guava colletotrichum orbiculare

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Effect test

Embodiment 1

[0025] Embodiment 1: PCR detects the design of primer sequence and the specific amplification of primer to guava anthracnose bacteria

[0026] 1. Design and synthesis of primers

[0027] The 10 guava anthracnose strains obtained by sequencing in our laboratory ( C. glosoporioides ) ITS sequence with GenBank Colletotrichum The ITS gene sequences of 18 different species of the genus were compared and analyzed for homology. According to the difference sites between the guava anthracnose bacteria and other species (compared in BioEdit), the PrimerPrimer5 software was used to design the guava anthracnose bacteria. C. glosoporioides Specific primers, the primer sequence is: upstream primer CGF: 5'-CGGGTAGGGTCTCCGTGA-3', downstream primer CGR: 5'-CCCAGTGCGAGACGTAAAGT-3', the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0028] Extraction of genomic DNA of tested strains

[0029] Genomic DNA of the tested strain was extracted by the CTAB method. Th...

Embodiment 2

[0034] Embodiment 2: Sensitivity detection of primers to the genomic DNA of guava anthracnose bacteria

[0035] 1. Conventional PCR amplification

[0036] The genomic DNA of the guava anthracnose bacteria was diluted with sterile ultrapure water, and prepared into a series of 10-fold concentrations for future use. Use the primer CGF / CGR of the present invention to carry out PCR amplification to the genomic DNA of different series concentrations, evaluate the sensitivity of this primer to the detection of guava anthracnose bacterial genomic DNA, amplification reaction system and reaction procedure are as follows: PCR reaction system 25 μ L, Including 2.5 μL 10×PCRbuffer (Mg 2+ free), 2.0mmol / LMgCl 2 , 0.2mmol / LdNTP, 1.0U Taq DNA polymerase (Takara Dalian Treasure Bioengineering Co., Ltd.), primers CGF / CGR 0.4 μmol / L each, 25ng DNA template, and the insufficient part was made up with sterile ultrapure water. The amplification reaction program was: pre-denaturation at 94...

Embodiment 3

[0040] Embodiment 3: the detection of guava anthracnose bacteria in diseased fruit

[0041] Extraction of Guava Anthracnose Fungus DNA from Diseased Fruits : DNA was extracted by NaOH rapid lysis method, the specific process is as follows: add 0.5mol / L NaOH 10μL to 1.0mg of diseased fruit, grind the tissue fully into a paste, transfer it to a 1.5mL centrifuge tube, centrifuge at 12,000rpm for 6min, and take the supernatant Add 0.1mol / L Tris-HCl (pH=8.0) 495μL to 5μL and mix well, take 1.0μL as PCR template for amplification. PCR amplification detection: The primer CGF / CGR of the present invention is used for PCR amplification. PCR reaction system 25 μL, including 2.5 μL 10×PCRbuffer (Mg 2+ free), 2.0mmol / LMgCl 2 , 0.2mmol / LdNTP, 1.0U Taq DNA polymerase (Takara Dalian Treasure Bioengineering Co., Ltd.), primers CGF / CGR 0.4 μmol / L each, 25ng DNA template, the insufficient part was made up with sterile ultrapure water; amplification parameters were: 95°C pre-denaturation...

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Abstract

The invention provides a guava colletotrichum orbiculare specificity PCR detecting primer and a detecting method of guava colletotrichum orbiculare. The primer comprises an upstream primer body CGF:5'-CGGGTAGGGTCTCCGTGA-3'and a downstream primer body CGR:5'-CCCAGTGCGAGACGT AAAGT-3', the PCR detecting method of guava colletotrichum orbiculare is built on the basis of the primer, and an amplification product with the fragment of 390 bp can be specifically amplified in guava colletotrichum orbiculare pure DNA and diseased tissue with guava colletotrichum orbiculare by agarose gel electrophoresis. When the detecting primer and the detecting method are used for detecting guava colletotrichum orbiculare, the advantages of being high in accuracy, high in specificity, high in sensitivity, simple and fast in detecting process operation are achieved, the detecting primer and the detecting method can be used for early diagnosis of guava anthracnose and monitoring and authenticating of germs in fields, and reliable technological and theoretical foundations are provided for prevention and treatment of guava anthracnose.

Description

technical field [0001] The invention relates to specific PCR detection primers for guava anthracnose bacteria and a detection method thereof, which are specially used for rapid, sensitive and specific molecular detection of guava anthracnose bacteria, and can be used for early diagnosis of guava anthracnose in the field and monitoring and identification of bacteria. The invention belongs to the technical field of detection, identification and control of crop diseases. Background technique [0002] Glosospora anthracnose ( Colletotrichum losoporioides ) infection caused by guava anthracnose is an important pre-harvest and post-harvest disease of guava in tropical and subtropical regions of the world. Around the world, the annual loss of guava due to this disease reaches 5% to 15%. Guava is usually infected with the disease in the early stage of ripening, but the pathogen remains dormant until the fruit matures before the development of disease symptoms. The initial symptom o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/645
CPCC12Q1/6895C12Q2600/166
Inventor 兰成忠姚婂爱余德亿阮宏椿黄鹏
Owner INST OF PLANT PROTECTION FAAS
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