Kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) and detection method

A technology of enterobacteriaceae and carbapenems, applied in the field of detection of carbapenem-resistant enterobacteriaceae, can solve the problems of high detection cost, low detection throughput, and poor integration level, and achieve simple operation, good effect, and good Effects of Specificity and Efficiency

Inactive Publication Date: 2016-06-15
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are many types of methods currently used for CRE genotype detection, including PCR method, loop-mediated isothermal amplification method, gene chip, etc. These methods have problems such as low detection throughput, poor integration, and relatively high detection costs.

Method used

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  • Kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) and detection method
  • Kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) and detection method
  • Kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 is used for the preparation of carbapenem-resistant Enterobacteriaceae (CRE) detection kit

[0076] The kit for detecting carbapenem-resistant Enterobacteriaceae (CRE) provided by the present invention consists of the following:

[0077] 1. PCR reaction mixture

[0078] The components and concentrations of the PCR reaction mixture are as follows: 8 μl of Buffer10×buffer solution, pair of detection primers, 0.3 μM of detection probe, and 0.8 mM of each component of dNTP;

[0079] The upstream and downstream detection primers in the detection primer pair are 0.3 μM respectively.

[0080] Detection primer pairs include all of the following detection primer pairs:

[0081] Detection primer pair 1:

[0082] KPC upstream primer is: 5'-ATCGCCGTCTAGTTCTGCTG-3', as shown in SEQ ID NO.1,

[0083] KPC downstream primer is: 5'-TATCCATCGCGTACACACCG-3', as shown in SEQ ID NO.2;

[0084] Detection primer pair 2:

[0085] NDM upstream primer is: 5'-GCAAATGGAAACTGGCGA...

Embodiment 2

[0122] Embodiment 2 is used to detect the use method of carbapenem-resistant Enterobacteriaceae (CRE) kit

[0123] 1. Reaction system preparation

[0124] components

volume

PCR reaction mix

34.4μL

Taq enzyme

0.6μL

sample DNA

5μL

total capacity

40μL

[0125] Take 34.4 μL of PCR reaction mixture, 0.6 μL of Taq enzyme and 5 μL of sample DNA and mix to form 40 μL of reaction solution, vortex and shake evenly, and then centrifuge.

[0126] 2. Fluorescence real-time quantitative PCR reaction and detection

[0127]

[0128] The fluorescent quantitative PCR reaction conditions are shown in the above table: the first stage: 94°C for 5 minutes; the second stage: 94°C for 20s, 58°C for 30s, 40 cycles, and the FAM / HEX signal was collected.

[0129] 3. Result judgment

[0130] After the reaction is over, the amplification curve and cycle number Ct of the samples in each reaction chamber are used as the basis for judging...

Embodiment 3

[0132] Example 3. Sensitivity and specificity analysis for detecting carbapenem-resistant Enterobacteriaceae (CRE) kit

[0133] 1. Preparation of reference DNA nucleic acid

[0134] Construct plasmids containing KPC, NDM, IMP, VIM or OXA-48 genes respectively

[0135] Using KPC, NDM, IMP, VIM or OXA-48 to be tested as templates, each standard product (SEQ ID NO.16-SEQ ID NO.20) as described in the summary of the invention was synthesized by Nanjing GenScript Biotechnology Company. Each standard was connected to the pUC57 vector and sequenced for proofreading.

[0136] The recombinant plasmid containing the KPC gene was named pUC57-PKC; the recombinant plasmid containing the NDM gene was named pUC57-NDM; the recombinant plasmid containing the IMP gene was named pUC57-IMP; the recombinant plasmid containing the VIM gene was named pUC57-VIM; The recombinant plasmid of -48 gene was named pUC57-OXA-48.

[0137] 2. Sensitivity and specificity analysis of the kit for detecting car...

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Abstract

The invention discloses a kit and a detection method for detecting carbapenem-resistant enterobacteriaceae (CRE). The detection primer pair in the present invention is selected from one or several pairs of KPC detection primer pair, NDM detection primer pair, IMP detection primer pair, VIM detection primer pair and OXA-48 detection primer pair. The kit described in the present invention covers common carbapenem-resistant genes, with a minimum detection of 1 copy/uL. The detection method of the invention has the characteristics of good sensitivity, high specificity, accurate quantification, etc., and has important clinical significance for the detection and monitoring of the increasingly severe CRE epidemic outbreak.

Description

technical field [0001] The invention belongs to the technical field of detection of carbapenem-resistant enterobacteriaceae (CRE), and in particular relates to a detection kit for carbapenem enzyme genes KPC, NDM, IMP, VIM and OXA-48 and a detection method thereof. Background technique [0002] Carbapenem antibiotics are atypical β-lactam antibiotics with the broadest antibacterial spectrum and the strongest antibacterial activity. Because of their stability to β-lactamase and low toxicity, they have become the most important treatment for severe bacterial infections. One of the antibacterial drugs. With the wide application of carbapenem antibiotics in clinic, serious drug resistance has emerged, and drug-resistant bacteria show extensive drug resistance. Before 2000, there were no widespread reports of carbapenemase, but more reports of carbapenem resistance caused by the loss of AmpC enzyme or ESBL combined with porin; in 2001, KPC was first reported in North Carolina, U...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2531/113C12Q2563/107C12Q2537/143
Inventor 张良禄殷雷洪昊岩
Owner WUHAN AIMISEN LIFE TECH CO LTD
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