Primer pair, probe and kit for detecting bacterium MCR-1 gene

A technology of MCR-1 and primer pairs, which is applied in the field of probes and kits and primer pairs to detect bacterial MCR-1 drug resistance genes, can solve the problems of primers and probe kits that do not yet exist, and achieve accurate detection results

Inactive Publication Date: 2016-03-23
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there are no primers, probe

Method used

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  • Primer pair, probe and kit for detecting bacterium MCR-1 gene
  • Primer pair, probe and kit for detecting bacterium MCR-1 gene
  • Primer pair, probe and kit for detecting bacterium MCR-1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 is used for detecting the kit of bacterial MCR-1 drug resistance gene and preparation method thereof

[0050] The kit for detecting bacterial MCR-1 drug resistance gene provided by the present invention consists of the following:

[0051] PCR reaction mix

[0052] The components and concentrations of the PCR reaction mixture are as follows: Buffer10×buffer 8 μL, upstream primer for detecting MCR-1 gene 0.6 μM, downstream primer for detecting MCR-1 gene 0.6 μM, MCR-1 probe 0.3 μM, amplification The upstream primer of the E.coli standard is 0.6 μM, the downstream primer of the amplified E.coli standard is 0.6 μM, the internal control probe is 0.3 μM, and each component of dNTP is 0.8 mM.

[0053] Wherein, in a specific embodiment, Buffer10× buffer is 10× ExTaqBuffer (Mg 2+ plus), 10×ExTaqBuffer (Mg 2+ plus) buffer was purchased from TAKARA Bao Biological Engineering (Dalian) Co., Ltd., model 9152A.

[0054] The upstream primer sequence for detecting the M...

Embodiment 2

[0063] Example 2 is used to detect the use method of the kit of bacterial MCR-1 drug resistance gene

[0064] 1. Sample DNA extraction:

[0065] The kit used is the OMEGA Bacterial Genome Extraction Kit. For specific steps, please refer to the instruction manual. The contents of the instruction manual are as follows:

[0066] (1) Take 1 mL of bacterial culture solution or bacteria, and centrifuge at 10,000×g for 1 min.

[0067] (2) Remove the supernatant, add 250 μL solution I (containing RNaseA), and vortex until the cells are completely suspended.

[0068] (3) Add 250 μL of solution II, and gently invert the centrifuge tube 4-6 times to obtain a clear lysate. It is best to incubate at room temperature for 2 minutes. Vigorous mixing will shear the chromosomal DNA and reduce the purity of the plasmid. (Store solution II should be tightly capped).

[0069] (4) Add 350 μL of solution III, mix gently by inverting several times until white flocculent precipitates appear, and c...

Embodiment 3

[0089] Example 3. Sensitivity analysis for detecting bacterial MCR-1 drug resistance gene kit

[0090] 1. Preparation of reference DNA nucleic acid

[0091] Construction of a plasmid containing the MCR-1 gene

[0092] The MCR-1 gene (SEQ ID NO.7) as described in the summary of the invention was synthesized by GenScript. The synthetic gene was connected to the pUC57 vector (provided by GenScript), a plasmid containing pUC57-MCR1 was constructed, and sequenced for proofreading.

[0093] 2. Sensitivity and specificity analysis of the kit for detecting MCR-1 drug resistance gene

[0094] Preparation of reference substances at different concentrations

[0095] Quantify the plasmid containing the MCR-1 gene synthesized in the above steps, and quantify it as 3×10 10 copies / μL, and serially diluted to obtain 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, 3×10 1 copies / μL, 3×10 0 Copy / μL and other different concentration gradient dilutions.

[0096] ...

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Abstract

The invention relates to a primer pair, a probe and a kit for detecting the bacterium MCR-1 drug-resistant gene. The primer pair comprises an MCR-1 gene detecting upstream primer including the sequence shown by SEQ ID NO.1 and an MCR-1 gene detecting downstream primer including the sequence shown by SEQ ID NO.2. The probe for the MCR-1 gene comprises a sequence shown by SEQ ID NO.3, a fluorescence group is integrated at the 5' end, and a quenching group is integrated at the 3' end of the probe for the MCR-1 gene. The kit for detecting the bacterium MCR-1 drug-resistant gene comprises the primer pair and the probe. The primer pair, the probe and the kit for detecting the bacterium MCR-1 drug-resistant gene have the advantages of being capable of detecting the bacterium MCR-1 drug-resistant gene, good in sensitivity, high in specificity, accurate in rationing and the like. The primer pair, the probe and the kit are good for clinical detection and monitoring of the bacterium MCR-1 drug-resistant gene and guiding antibiotic therapy of patients.

Description

technical field [0001] The invention belongs to the technical field of antibiotic resistance gene detection, and in particular relates to a primer pair, a probe and a kit for detecting bacterial MCR-1 drug resistance gene. Background technique [0002] Since the 1920s, a large number of natural antibiotics including penicillin and streptomycin have been discovered one after another, thus human beings have opened the era of antibiotics and have an absolute advantage in the fight against bacteria. However, with the over-reliance on the use of antibiotics, the problem of bacterial resistance has become increasingly prominent. The abuse of antibiotics has screened and enriched randomly mutated drug-resistant bacteria, which has weakened or even invalidated the power of antibiotics from generation to generation. The war between humans and bacteria has gradually turned into a "protracted war" around the issue of "drug resistance". Polymyxins (Polymyxins) is a group of polypeptid...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02C12N15/11C12R1/19C12R1/445C12R1/22C12R1/145C12R1/36
CPCC12Q1/689
Inventor 张良禄殷雷洪昊岩
Owner WUHAN AIMISEN LIFE TECH CO LTD
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