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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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 c

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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

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Example Embodiment

[0058] Embodiment 1 The primer specificity detection of pectinobacter carotiorum

[0059] 28 strains of pectinobacterium carotii and 13 strains of common fungi and bacterial disease pathogens on other vegetables (as shown in Table 2) were respectively used to specifically detect the primer composition of pectin bacillus carotii. Among them, the pathogenic bacterial strains were cultivated in NA medium, and the pathogenic fungal strains were cultivated in PDA medium. Subsequently, the pathogenic bacteria were activated by NA medium and transferred to NB medium for 22-26h shaking culture at a temperature of 26-30°C and a rotation speed of 120-130rpm, and the bacterial suspension was collected to extract DNA. The pathogenic fungi were activated by PDA medium and transferred to PD medium. The mycelia were collected and DNA was extracted after 7 days of shaking culture at 26-30°C and a rotating speed of 120-130rpm. The DNA was extracted from the pathogenic bacteria using a DNA ext...

Example Embodiment

[0073] Embodiment 2 The primer sensitivity detection of pectinobacter carotovora

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

[0075] like figure 2 As shown, the detection results show that the LAMP detection technology can detect P. carotii subsp. carrot in the concentration range of 1.92 × 10 1 ng / μl-5.8×10 -3 The results of ng / μl were good, indicating that the detection sensitivity of the DNA samples of P. carotidum subsp. carotiensis strains reached 1.92×10 -3 ng / μl.

Example Embodiment

[0076] Embodiment 3 Detection of Carotid rot Pectinobacillus in diseased celery tissue samples

[0077] Sampling was performed on naturally occurring celery tissues (collected from fields in Beijing, Hebei, and Henan, respectively, number 1-3) and artificially inoculated celery tissues (number 4-10, as shown in Table 3). The celery stalk segment with disease symptoms, the disease symptoms include that the stalk is wet and rotten and turns black and smelly. At the same time, healthy celery tissue in the non-affected area was selected as a negative control sample (No. 11).

[0078] Table 3 Pathogens of artificial inoculation of celery hosts

[0079]

[0080] After the samples were collected, they were washed repeatedly, and the surface was disinfected with 75vol% ethanol, followed by repeated washing with sterile water, and the plant tissue was extracted and DNA was purified by using a 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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