LAMP detection method of clostridium difficile binary toxin and special primers and kit for LAMP detection method

A technology of Clostridium difficile and binary toxins, which is applied in the field of molecular biology detection of bacteria, can solve the problems of multiple DNA spectrum bands, easy contamination, and difficulty in distinguishing, and achieves broad market prospects, simple result identification, and simple operation. Effect

Inactive Publication Date: 2015-02-04
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Disadvantages: long typing time, complex electrophoresis conditions, special instruments and expensive reagents
Disadvantages: There are too many bands in the DNA map, it is difficult to distinguish, and it is easy to contaminate
But so far, there are no LAMP-specific primers and kits for the detection of Clostridium difficile binary toxins on the market.

Method used

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  • LAMP detection method of clostridium difficile binary toxin and special primers and kit for LAMP detection method
  • LAMP detection method of clostridium difficile binary toxin and special primers and kit for LAMP detection method
  • LAMP detection method of clostridium difficile binary toxin and special primers and kit for LAMP detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 implements the present invention through the following steps:

[0061] 1. Primer design for LAMP detection of Clostridium difficile binary toxin

[0062] (1) Primer design for LAMP detection of Clostridium difficile cdtA: The cdtA repeat sequence of Clostridium difficile (GenBank: HQ639673.1) was retrieved from the American Gene Database, and it was found to be Clostridium difficile through homology analysis by BLAST software Specific conserved sequence (SEQ ID NO: 1 in the sequence list), and then according to the conserved target DNA sequence, use the software Primer design V4 to design primers for LAMP detection of Clostridium difficile cdtA, and optimize the results to obtain three sets of primer pairs, F3 and B3 are the first group, FIP and BIP are the second group, LF and LB are the third group, and the sequences of specific primers are shown in Table 1.

[0063] Table 1 cdtA primers used for LAMP detection of Clostridium difficile

[0064]

[00...

Embodiment 2

[0075] Example 2 Under the same reaction system, the genomic DNA containing cdtA and cdtB Clostridium difficile 027 / B1 / NAP1 under different reaction conditions were detected by LAMP to obtain the optimal reaction temperature.

[0076] After testing, the turbidimeter detection result of the optimal temperature screening of the LAMP detection method of Clostridium difficile cdtA of the present invention (such as figure 1 shown): at 58-62°C, the reaction time fluctuates between 47-48min, and the best reaction condition is: 60°C constant temperature for 90min.

[0077] The turbidimeter detection result of the optimal temperature screening of the LAMP detection method of Clostridium difficile cdtB of the present invention (as figure 2 shown): at 61-65°C, the reaction time fluctuates between 35-40min, and the best reaction condition is: 63°C constant temperature for 90min. When Clostridium difficile cdtA and cdtB are detected at the same time, the optimal reaction condition is: co...

Embodiment 3

[0078] Example 3 Specific detection of the LAMP detection method of Clostridium difficile binary toxin of the present invention.

[0079] 1. The specificity detection of the LAMP detection method of Clostridium difficile cdtA 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 and Haemophilus influenzae type B (all the above strains are from the Center ...

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Abstract

The invention discloses an LAMP detection method of clostridium difficile binary toxin and special primers and kit for the LAMP detection method. The LAMP detection method comprises the following steps: designing the primers according to specificity conserved genes cdtA and cdtB of clostridium difficile, performing LAMP amplification under the guide of the obtained primers by taking the genome DNA of a sample to be tested as a template, and further rapidly and quantitatively detecting whether the sample to be tested carries the clostridium difficile binary toxin according to the color change of reaction liquid or the turbidity change of the reaction liquid. By adopting the LAMP detection method, clostridium difficile cdtA or/and cdtB can be independently or simultaneously detected, rapid, convenient, synchronous, efficient, high-specificity and high-sensitivity detection under an isothermal condition can be achieved without complex instruments, a novel technical platform can be provided for clostridium difficile binary toxin detection and toxin classification, clinical diagnosis and treatment can be instructed, fulminant diffusion of clostridium difficile high-toxicity strains can be prevented, the method can be used for screening and detecting the clostridium difficile binary toxin in primary medical treatment and public health departments and disease prevention and control centers and has a wide market prospect and relatively great economic and social benefits.

Description

technical field [0001] The invention relates to a LAMP detection method of Clostridium difficile binary toxin and its special primer and kit, belonging 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 referred to as Clostridium difficile infection (CDI). [0003] Clostridium difficile can be divided into toxin-producing strains and non-toxin-producing strains, and the non-toxin-producing strains do not cause clin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/145
CPCC12Q1/6844C12Q1/689C12Q2600/158C12Q2600/16C12Q2531/119
Inventor 陈烨林敏怡王浦谭嘉圣周有连袁静刘威
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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