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Method for detecting pectobacterium carotovorum based on loop-mediated isothermal amplification technology, primer combination of method and detection kit

A technology of carrot soft rot and primer composition, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problem of being unable to quickly detect carrot soft rot pectin bacillus and the consumption of biological detection methods. Long time, relying on thermal cycle equipment and other issues, to achieve the effect of low detection cost, fast identification speed, good specificity and sensitivity

Active Publication Date: 2015-11-04
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the defects of the prior art, for the defects of the existing biological detection method of pectin bacillus carotovora soft rot, such as time-consuming, complicated operation and poor accuracy, and must rely on the thermal cycler for the PCR detection technology, so it cannot The rapid detection of Pectinobacillus carotovora has the problem of difficulty in popularization. A new detection method for Pectinobacillus carotovora and specific primers and detection kits for realizing the method are provided.

Method used

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  • Method for detecting pectobacterium carotovorum based on loop-mediated isothermal amplification technology, primer combination of method and detection kit
  • Method for detecting pectobacterium carotovorum based on loop-mediated isothermal amplification technology, primer combination of method and detection kit
  • Method for detecting pectobacterium carotovorum based on loop-mediated isothermal amplification technology, primer combination of method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Primer Specific Detection of Carrot Pectin Bacillus

[0059] 28 strains of Pectinobacillus carotovora and 13 strains of common fungi and bacterial disease pathogens on other vegetables (as shown in Table 2) were used to detect the specificity of the primer composition of Pectinobacillus carotovora. Wherein, the pathogenic bacterial strains are cultivated with NA medium, and the pathogenic fungal strains are cultivated with PDA medium. Then the pathogenic bacteria are activated by the NA medium and then transferred to the NB medium, shaken and cultured for 22-26h at a temperature of 26-30°C and a rotational speed of 120-130rpm, and collect the bacterial suspension to extract DNA. The pathogenic fungi were activated by PDA medium and then transferred to PD medium, cultured with shaking at 26-30°C and rotating speed 120-130rpm for 7 days, and then the mycelia were collected to extract DNA. DNA extraction kits were used to extract DNA from pathogenic bacteria.

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Embodiment 2

[0073] Example 2 Primer Sensitivity Detection of Carrot Soft Rot Pectin Bacillus

[0074] The DNA of Pectinobacillus carotovora subsp. carotovii isolated from the celery host collected in Beijing in 2014 (No. QC14052001) was used as a template for the detection of primer sensitivity. After the target strain DNA was extracted, the concentration was measured, and then 10-fold serial dilution was prepared to prepare 1.92×10 1 ng / μl-1.92×10 -6 ng / μl, respectively numbered 1-8, and pure water control numbered 9, the LAMP reaction was carried out according to the reaction system and reaction process described in Example 1.

[0075] Such as figure 2 As shown, the detection results show that the LAMP detection technology can detect Pectinobacillus carotovora subspecies in the concentration range of 1.92×10 1 ng / μl-5.8×10 -3 The ng / μl result was good, indicating that the detection sensitivity of the DNA sample of Pectinbacterium carotovora subsp. carotovii reached 1.92×10 -3 ng / μ...

Embodiment 3

[0076] Example 3 Detection of Pectin Bacillus carotovora soft rot in diseased celery tissue samples

[0077] Samples were taken from naturally occurring celery tissues (collected from the fields of Beijing, Hebei, and Henan respectively, No. 1-3) and artificially inoculated celery tissues (No. 4-10, as shown in Table 3). The samples were samples with typical soft rot The celery stalks with symptoms of disease symptoms include wet rot and blackened and smelly stalks, and healthy celery tissues in non-infested areas were selected as negative control samples (No. 11).

[0078] Table 3 Pathogens artificially inoculated by celery host

[0079]

[0080] After the samples were collected, they were washed repeatedly, and 75vol% ethanol was used for surface disinfection, followed by repeated washing with sterile water. The plant tissue extraction and DNA purification were carried out using the spin column type plant genomic DNA extraction kit (purchased from Beijing Tiangen Company)...

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Abstract

The invention relates to a method for detecting pectobacterium carotovorum based on a loop-mediated isothermal amplification technology, a primer combination of the method and a detection kit. An LAMP primer set used for detecting the pectobacterium carotovorum is composed of a positive inner primer FIP, a negative inner primer BIP, a positive outer primer F3, a negative primer B3 and a loop primer LOOP. The primer combination and the detection kit are high in specificity and good in flexibility and are obviously superior to other detection means in the prior art. Compared with a traditional PCR, the detection method and the kit are visual in result, easy to operate and low in cost; detection of pectobacterium carotovorum in the total DNA in various plant morbidity tissues can be achieved, a pectobacterium carotovorum bacterial suspension can directly detected, and a foundation is laid for direct field diagnosis of diseases.

Description

【Technical field】 [0001] The field of gene detection and diagnosis of the present invention specifically relates to a method for detecting Pectinobacillus carotovora based on loop-mediated isothermal amplification technology, the primer composition involved, the application of the primer composition and the method for Pectinbacterium carotovora detection kits. 【Background technique】 [0002] Pectobacterium carotovorum is a worldwide epidemic disease that causes vegetable bacterial soft rot. Its host range is very wide and can cause a variety of horticultural crops such as celery, carrot, tomato, cucumber, potato, melon Soft rot occurs and brings huge economic losses. Due to the strong pathogenicity of Pectin Bacillus soft rot, it is difficult to control the disease once it occurs, and there is a lack of specific fungicides for bacterial soft rot in the market. Therefore, the prevention and control of this disease is mainly prevention. To prevent the occurrence of diseases,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 石延霞晋知文李宝聚谢学文柴阿丽
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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