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LAMP kit for VanB gene detection and primer special for same

A kit and gene technology, applied in the application of VanB gene detection, LAMP kit, the field of molecular biology detection of genes, can solve problems such as unseen, and achieve simple operation, short detection time and high amplification efficiency. Effect

Active Publication Date: 2015-12-23
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no LAMP kits and special primers for the detection of VanB gene in China

Method used

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  • LAMP kit for VanB gene detection and primer special for same
  • LAMP kit for VanB gene detection and primer special for same
  • LAMP kit for VanB gene detection and primer special for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Design of primers for LAMP detection of VanB gene

[0044] The VanB gene sequence (GenBank number: KC489784.1) was retrieved from NCBI's nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov), and the homology analysis was performed by BLAST software to obtain the specificity of VanB gene Conserved target sequence (SEQIDNO: 1 in the sequence list), and then according to the conservative target DNA sequence, use the software PrimerdesignV4 to design primers for LAMP detection of VanB gene, and design four sets of primers (codenamed VanB-1, VanB- 6. VanB-9, VanB-12). After experimental comparison, the primer combination VanB12 was finally selected. The primer sequence is as follows.

[0045] Table 1 Primer combination VanB-12 used for LAMP detection of VanB

[0046]

[0047]

Embodiment 2

[0048] Example 2. LAMP detection of VanB gene

[0049] 1. Determine the best primer combination for VanB gene LAMP detection

[0050] The four sets of primer combinations (codenamed VanB-1, VanB-6, VanB-9, VanB-12) designed in Example 1 were used for LAMP detection of VanB gene to obtain the best primer combination. The specific method is as follows:

[0051] 1) Extract the genomic DNA of Enterococcus V583

[0052] With Promega GenomicDNAPurificationKitA1125 extracts the genomic DNA of Enterococcus faecalis V583. The specific extraction method includes the following steps:

[0053] 1. Pipet 560μL of prepared bufferAVL (containing carrierRNA) into a 1.5mL centrifuge tube. (Adjust bufferAVL-carrierRNA proportionally to the actual amount of the sample)

[0054] 2. Add 140μL of plasma, serum, urine, cultured cell supernatant or cell-free body fluid to the centrifuge tube containing bufferAVL-carrierRNA. Mediate for 15 seconds and mix well.

[0055] 3. Leave at room temperature for 10 minut...

Embodiment 3

[0074] Example 3. Detection of specificity and sensitivity of the LAMP detection method of VanB gene of the present invention

[0075] 1. Specific detection of the LAMP detection method of VanB gene of the present invention

[0076] Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis, Vibrio cholerae O139 group, Bacillus anthracis, Escherichia coli, Enterohemorrhagic Escherichia coli, Yersinia enterocolitica , Salmonella typhimurium, Salmonella aberdeen, Listeria monocytogenes, Vibrio parahaemolyticus, Acinetobacter rouxii, Enteropathogenic Escherichia coli, Klebsiella pneumoniae, Chronicles Freundii Gagella (from the Infectious Disease Control Center of the Institute for Disease Control and Prevention of the Academy of Military Medical Sciences), Enterococcus faecalis V309 carrying the VanA gene, Enterococcus faecalis-1, Enterococcus faecalis-2, Enterococcus faecalis-3, Enterococcus faecalis-4 , Enterococcus faecium-1, Enterococcus fae...

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Abstract

The invention discloses a LAMP kit for VanB gene detection and a primer special for the same. The LAMP primer for VanB gene detection is designed according to a specific conservative target sequence of a VanB gene and consists of four primer bodies including outer primers VanB12-F3 and VanB12-B3 and inner primers VanB12-FIP and VanB12-BJP. The VanB genes in pure bacteria, patients' faeces, blood and other samples can be qualitatively detected under the isothermal condition in a fast, convenient, efficient, high-specificity and high-sensitivity mode without complex apparatuses, and a new technical platform is provided for VanB gene detection.

Description

Technical field [0001] The invention belongs to the molecular biology detection of genes in the field of biotechnology, and particularly relates to a LAMP kit of VanB gene and its special primers and its application in the detection of VanB gene. Background technique [0002] VanB gene is one of the resistant genotypes of Vancomycin-resistant Enterococci (Vancomycinresistant Enterococci, VRE), which mainly exists in Enterococcus faecalis and Enterococci faecium. VanB phenotypic drug resistance genes are mostly located on the host chromosome, and can also be present on plasmids. Through the conjugation mechanism, the drug resistance genes can be spread horizontally or vertically within or between species and transmitted to other enterococci. Genus or other bacteria. [0003] Vancomycin, a glycopeptide antibiotic discovered in the 1950s, was used as the first choice for the treatment of multi-drug resistant G+ strain infections. It was once hailed as "human beings' last line of defe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 袁静李环赵向娜王雪松魏晓林维石
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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