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Lamp kit for detecting vanb gene and its special primers

A kit and gene technology, applied in the detection of VanB gene, LAMP kit, the field of molecular biology detection of genes, can solve the problems that have not been seen, and achieve the effect of simple operation, high sensitivity and good specificity

Active Publication Date: 2018-12-07
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 detecting vanb gene and its special primers
  • Lamp kit for detecting vanb gene and its special primers
  • Lamp kit for detecting vanb gene and its special primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Primers designed 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 specificity of the VanB gene was obtained through homology analysis by BLAST software The conserved target sequence (SEQ ID NO: 1 in the sequence table), and then according to the conserved target DNA sequence, use the software Primer design V4 to design primers for the detection of VanB gene by LAMP, and design four sets of primers (code names are respectively VanB- 1. VanB-6, VanB-9, VanB-12), after experimental comparison, the primer combination VanB12 was finally selected, and the primer sequence is as follows.

[0045] Table 1 Primer combination VanB-12 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] Carry out LAMP detection to VanB gene with four sets of primer combinations (code-named VanB-1, VanB-6, VanB-9, VanB-12) designed in Example 1 to obtain the best primer combination, the specific method is as follows:

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

[0052] Use Promega Genomic DNA Purification Kit A1125 extracts the genomic DNA of Enterococcus faecalis V583, and the specific extraction method includes the following steps:

[0053] 1. Pipette 560μL of prepared buffer AVL (containing carrier RNA) into a 1.5mL centrifuge tube. (Adjust the buffer AVL-carrier RNA proportionally according to the actual amount of the sample)

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

[0055...

Embodiment 3

[0074] Embodiment 3, specificity and sensitivity detection of the LAMP detection method of VanB gene of the present invention

[0075] One, the 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, enterohaemorrhagic Escherichia coli, Yersinia enterocolitica , Salmonella typhimurium, Salmonella Aberdeen, Listeria monocytogenes, Vibrio parahaemolyticus, Acinetobacter ruckeri, enteropathogenic Escherichia coli, Klebsiella pneumoniae, Chi flexneri Shigella (from Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences), Enterococcus faecalis V309 carrying VanA gene, Enterococcus faecalis-1, Enterococcus faecalis-2, Enterococcus faecalis-3, Enterococcus faecalis-4 , Enterococcus faecium-1, Enterococcus faecium-2, En...

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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 biological detection of genes in the field of biotechnology, in particular to a LAMP kit of VanB gene and its special primer and its application in detection of VanB gene. Background technique [0002] VanB gene is one of the drug-resistant genotypes of Vancomycin-resistant Enterococci (VRE), mainly present in Enterococcus faecalis and Enterococcus faecium. The VanB phenotype drug resistance gene is mostly located on the host chromosome, and can also exist on the plasmid. Through the conjugation mechanism, the drug resistance gene can be transmitted horizontally or vertically within or between species through the conjugation mechanism, and then passed to other enterococci or other bacteria. [0003] Vancomycin, a glycopeptide antibiotic discovered in the 1950s, was used as the drug of choice for the treatment of multidrug-resistant G+ strain infections, and was once known as "the last line of defense for humans a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12N15/11
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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