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A multiplex fluorescent quantitative PCR detection kit for vancomycin-resistant enterococci and its detection method

A multiplex fluorescence quantification, vancomycin intestinal technology, applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as difficulty in anti-infection treatment, and achieve quantitative detection results

Active Publication Date: 2016-05-11
BEIJING T&OINTL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, vancomycin-resistant enterococci have become an important monitoring object for clinical nosocomial infections. Once vancomycin-resistant enterococci break out and spread, it will bring great difficulties to anti-infection treatment.

Method used

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  • A multiplex fluorescent quantitative PCR detection kit for vancomycin-resistant enterococci and its detection method
  • A multiplex fluorescent quantitative PCR detection kit for vancomycin-resistant enterococci and its detection method
  • A multiplex fluorescent quantitative PCR detection kit for vancomycin-resistant enterococci and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A multiplex fluorescent quantitative PCR detection kit for vancomycin-resistant enterococcus described in the embodiments of the present invention, said kit includes VanA-specific primers, VanB-specific primers, VanA fluorescent probes, VanB fluorescent probes, PCR Buffer, deoxynucleoside triphosphate mixture, DNA polymerase, VanA gene, VanB gene, VanA gene standard, VanB gene standard.

[0052] The VanA-specific primers are as follows: VanA upstream primer: 5'-ttgacttcgttcagtac-3'

[0053] VanA downstream primer: 5′-ggagcgaggacggatacagga-3′

[0054] The VanA fluorescent probe is as follows: 5'-FAM-ctagacctctacagccga-BHQ1-3'

[0055] The VanB-specific primers are as follows: VanB upstream primer: 5'-ggatcaaatccggttgagc-3'

[0056] VanB downstream primer: 5′-tgtctgctggaacgataatca-3′

[0057] The VanB fluorescent probe is as follows: 5'-VIC-ccgcatccatcaggaa-BHQ1-3'.

[0058] The VanA gene standard product sequence is as follows: ttgacttcgttcagtacaatgcggccgttatcttgtaaa...

Embodiment 2

[0060] Embodiment 2: the acquisition of VanA and VanB gene standard

[0061] 1), material:

[0062] The pGEM-T-Easy cloning system, PCR-related reagents and TaqDNA polymerase were purchased from Promega, USA, 377 sequencer (ABI Company), Bio-Radicycler PCR instrument (Bio-Rad Company), and ABI7500fast quantitative PCR instrument (ABI Company).

[0063] 2), design and synthesis of specific primers and probes:

[0064] Using the Vancomycin-resistant Enterococcus VanA (GenBank registration number JN207930.1) and VanB (GenBank registration number U00456.1) genes as templates, use PrimerExpressTM (V3.0, American ABI Company) software to analyze TaqManTaqMan specific primers and Probe sites from which the best combination is selected. The primers and probes were synthesized and purified by Shanghai Huirui Biotechnology Co., Ltd.

[0065] 3), the preparation of VanA and VanB gene standards:

[0066] Using the Enterococcus standard strain (ATCC29212) as a template, extract vancomy...

Embodiment 3

[0075] Example 3: Method for detecting vancomycin-resistant enterococcus genes using the multiplex fluorescent quantitative PCR method of the kit

[0076] Described detection method comprises the steps:

[0077] 1) PCR amplification reaction of VanA gene standard substance and VanB gene standard substance:

[0078] Wherein, the PCR reaction solution is composed as follows:

[0079]

[0080] (dATP, dTTP, dCTP, dGTP substance ratio 1:1:1:1)

[0081] Gene standard DNA (VanA and VanB each 50ng / μL) 4μL

[0082] Make up to 50 μL with water.

[0083] The PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes, 40 cycles of amplification at 95°C for 15 seconds, and 45 seconds at 60°C, and finally at 4°C. Fluorescence acquisition is performed at the annealing temperature of each cycle;

[0084] 2) Draw a standard curve

[0085] Follow step 1) to test the VanA gene standard and VanB gene standard with different concentrations of 5ng / μL, 0.5ng / μL, 50pg / μL, and 5pg / μL ...

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Abstract

The invention relates to a vancomycin-resistant enterococcus multiplex fluorescence quantitative PCR (polymerase chain reaction) detection kit. The kit comprises a VanA specific primer, a VanB specific primer, a VanA fluorescent probe, a VanB fluorescent probe, a PCR buffer solution, a deoxynucleoside triphosphate mixture, a DNA (deoxyribonucleic acid) polymerase, a VanA gene standard product sequence and a VanB gene standard product sequence. The kit disclosed by the invention has the beneficial effects that due to the adoption of the PCR detection method, accurate, fast and quantitative detection can be realized.

Description

technical field [0001] The invention relates to a multiplex fluorescent quantitative PCR kit for vancomycin-resistant enterococci and a detection method thereof. Background technique [0002] Vancomycin Resistant Enterococcus (VancomycinResistantEnterococcus) was first isolated in France in 1986. Since then, other European countries and the United States have successively detected and isolated strains of vancomycin-resistant Enterococcus strains, which have become important pathogenic bacteria for nosocomial infections. Vancomycin-resistant enterococci are often associated with high morbidity and mortality due to severely limited drug options. Therefore, vancomycin-resistant enterococcus has become an important monitoring object for clinical nosocomial infection. Once vancomycin-resistant enterococcus breaks out and spreads, it will bring great difficulties to anti-infection treatment. [0003] Over the past two decades, vancomycin-resistant enterococci have seen a trend t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6851C12Q2537/143C12Q2545/113C12Q2563/107
Inventor 程伟金大智
Owner BEIJING T&OINTL TECH
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