Lamp detection method of Clostridium difficile ab toxin and its special primers and kit

A Clostridium difficile and kit technology, applied in the field of molecular biology detection of bacteria, can solve problems such as cumbersome interpretation methods, unfavorable Clostridium difficile toxin typing, and inability to quickly interpret results with the naked eye, achieving high-efficiency amplification and results The effect of easy identification and simple operation

Inactive Publication Date: 2016-07-13
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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Problems solved by technology

In 2005, Haru Kato et al. (Kato, H. and T. Yokoyama, et al. (2005). Rapid and simple method for detecting the toxin B gene of Clostridium difficile ins tool specimens by loop-mediated disothermal amplification. JClin Microbiol 43 (12): 6108-12.) proposed to use the LAMP method to design 6 strains for the detection of the tcdB difficile gene. , interpret the results by turbidimeter and polyacrylamide gel electrophoresis, the test results are specific, sensitive, and fast, but the disadvantage is that it cannot detect whether Clostridium difficile carries the tcdA gene, which is not conducive to the toxin typing of Clostridium difficile, and the result interpretation method is cumbersome , Relevant instruments are needed, and it is impossible to quickly interpret the results with the naked eye
Liu Chang et al. in 2012 (Liu Chang, Jiang Dongneng, Pu Xiaoyun. Research on rapid detection of Clostridium difficile in feces [J]. International Journal of Laboratory Medicine. 2012 (20)) and 2013 (Liu, C. and D.N. Jiang , etal. (2013). "DNA detection of Clostridium difficile infection based on real-time resistance measurement." GenetMolRes12 (3): 3296-304.) proposed: 4 primers were designed for the tcdA gene to detect C. The method is specific, sensitive, and fast, and is suitable for on-site and grassroots promotion. The disadvantage is that it cannot detect the carrying status of Clostridium difficile tcdB
At present, there is a lack of specific primers designed for the tcdA gene and tcdB gene of Clostridium difficile, and the fast, simple, specific and sensitive simultaneous detection of Clostridium difficile tcdA and tcdB can be achieved by reading the results with chromogenic reagents

Method used

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  • Lamp detection method of Clostridium difficile ab toxin and its special primers and kit
  • Lamp detection method of Clostridium difficile ab toxin and its special primers and kit
  • Lamp detection method of Clostridium difficile ab toxin and its special primers and kit

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Embodiment 1

[0059] Embodiment 1 implements the present invention through the following steps:

[0060] 1. Primer design for LAMP detection of Clostridium difficile

[0061] (1) Primer design for LAMP detection of Clostridium difficile tcdA: The repetitive sequence of Clostridium difficile tcdA (GenBank: X92982.1) was retrieved from the American Gene Database, and it was found to be Clostridium difficile through homology analysis by BLAST software Specific conserved sequence (SEQIDNO: 1 in the sequence list), and then according to the conserved target DNA sequence, use the software PrimerdesignV4 to design primers for LAMP detection of Clostridium difficile. As a result, three sets of primer pairs were optimized, and F3 and B3 were the first group, FIP and BIP are the second group, LF and LB are the third group, and the specific primer sequences are shown in Table 1.

[0062] Table 1 tcdA primers used for LAMP detection of Clostridium difficile

[0063]

[0064] (2) Primer design for ...

Embodiment 2

[0070] Example 2 Optimum temperature screening experiment of the Clostridium difficile LAMP detection method of the present invention: Using the primers and amplification reaction system of Example 1, under the same reaction system, the genomic DNA of Clostridium difficile under different reaction conditions (temperature) was carried out LAMP detection to obtain the optimal reaction temperature for the amplification reaction.

[0071] After testing, the turbidimeter detection result of the optimal temperature screening of the LAMP detection method of Clostridium difficile tcdA of the present invention (such as figure 1 shown): the optimal temperature under the reaction condition of 58-69°C for 60 minutes is 61°C. The turbidimeter detection result of the LAMP detection method optimal temperature screening of Clostridium difficile tcdB of the present invention (as figure 2 Shown): The optimal temperature is 60°C under the reaction condition of 58-69°C constant temperature for...

Embodiment 3

[0072] Example 3 Specific detection of the LAMP detection method for Clostridium difficile of the present invention.

[0073] 1. The specificity detection of the LAMP detection method of Clostridium difficile tcdA of the present invention: Bacillus megaterium, Vibrio sharkus, Pseudomonas maltophilia, Mycobacterium tuberculosis, Vibrio cholerae O139 group, Bacillus anthracis, enterohemorrhagic large intestine Bacillus, Yersinia enterocolitica, Vibrio parahaemolyticus, Enteropathogenic Escherichia coli, Enteroadhesive Escherichia coli, Enteroinvasive Escherichia coli, Enterotoxigenic Escherichia coli, Yersinia pestis, Pneumonia Streptococcus, Neisseria meningitidis, Burkholderia pseudomallei, Methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Bordetella pertussis, Haemophilus influenzae, Corynebacterium diphtheriae, Genomic DNA of Pseudomonas aeruginosa, Haemophilus influenzae type B, and Acinetobacter brucei (all the above strains are from t...

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Abstract

The invention discloses an LAMP detection method for clostridium difficile AB toxins and a special primer and a kit thereof. The LAMP detection method disclosed by the invention comprises the following steps: designing a primer according to the specific conserved genes tcdA and tcdB of clostridium difficile; then, by taking a genomic DNA of an object to be detected as a template, carrying out LAMP amplification under the guide of the obtained primer; and synchronously and qualitatively detecting toxigenic strains of clostridium difficile in a sample to be detected through the color changes and turbidity changes of a reaction liquid. According to the invention, toxigenic strains of clostridium difficile can be detected in a fast, convenient, synchronous, high efficiency, high specificity and high sensitivity mode under isothermal conditions without using complex instruments, so that a new technical platform is provided for the detection and toxin classification of clostridium difficile, therefore, the LAMP detection method disclosed by the invention can be used for screening and detecting clostridium difficile by grassroots medical health units and various disease prevention and control centers, has a broad market prospect and great economic and social benefits, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to a LAMP detection method of Clostridium difficile AB toxin, a special primer and a kit thereof, and belongs to the technical field of bacterial molecular biology detection methods in the field of biotechnology. Background technique [0002] Clostridium difficile (Cd) is a Gram-positive anaerobic bacillus that is widely distributed in water, soil and other natural environments, as well as animal and human feces. Clostridium difficile is an opportunistic pathogenic bacterium that is not invasive itself. When the normal microenvironment of the human intestinal tract is damaged, some toxin-producing bacteria can cause antibiotic-associated diarrhea, colonic Inflammation and even fatal pseudomembranous colitis, collectively known as Clostridium difficile infection (Clostridium difficile infection, CDI). Among the pathogens identified in hospital-acquired infectious diarrhea, Clostridium difficile is the most common; among the causes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/145
CPCC12Q1/6844C12Q1/689C12Q2600/16C12Q2531/119C12Q2537/143
Inventor 陈烨林敏怡王浦谭嘉圣张婷袁静刘威
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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