Fluorescent quantitative PCR detection method for drug resistance gene mcr (mobile colistin resistance)-4/5/8

A technology of drug resistance genes and primer pairs, which can be used in microorganism-based methods, biochemical equipment and methods, and determination/inspection of microorganisms, which can solve problems such as time-consuming and labor-intensive

Active Publication Date: 2018-11-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, traditional detection methods, such as conventional PCR and Sanger sequencing, are time-consuming and labor-intensive
There is currently no real-time PCR detection method for mcr-4 / 5 / 8

Method used

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  • Fluorescent quantitative PCR detection method for drug resistance gene mcr (mobile colistin resistance)-4/5/8
  • Fluorescent quantitative PCR detection method for drug resistance gene mcr (mobile colistin resistance)-4/5/8
  • Fluorescent quantitative PCR detection method for drug resistance gene mcr (mobile colistin resistance)-4/5/8

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, design and preparation of primers

[0062] A large number of sequence analyzes and comparisons were carried out to obtain several primers for detecting the drug resistance gene mcr-4 / 5 / 8. Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally a primer set for detecting the drug resistance gene mcr-4 / 5 / 8 was obtained, as shown in Table 1.

[0063] Table 1 Primer information table of mcr-4 / 5 / 8 gene

[0064]

[0065] Primer F1 and primer R1 form primer pair I;

[0066] Primer F2 and primer R2 constitute primer pair II;

[0067] Primer F3 and primer R3 constitute primer pair III.

Embodiment 2

[0068] Embodiment 2, the construction of plasmid standard

[0069] 1. mcr-4 plasmid standard product: insert the double-stranded DNA molecule shown in sequence 7 of the sequence table into the EcoR V site of pDM19-T vector to obtain the mcr-4 plasmid standard product.

[0070] 2. mcr-5 plasmid standard product: insert the double-stranded DNA molecule shown in Sequence 8 of the sequence table into the EcoR V site of pDM19-T vector to obtain the mcr-5 plasmid standard product.

[0071] 3. mcr-8 plasmid standard product: insert the double-stranded DNA molecule shown in sequence 9 in the sequence table into the EcoRV site of pDM19-T vector to obtain the mcr-8 plasmid standard product.

Embodiment 3

[0072] Embodiment 3, primer verification and specificity experiment

[0073] 1. Plasmid Standard Verification Primers

[0074] Samples to be tested: 3 plasmid standards prepared in Example 2.

[0075] Using the sample to be tested as a template, PCR amplification was performed using primer pair I to primer pair III in Example 1, respectively.

[0076] PCR amplification reaction system (20 μl): upstream primer 0.8 μl, downstream primer 0.8 μl, 2×Taq PCRMasterMix (purchased from Nanjing Novizan Biotechnology Co., Ltd., P111-01) 10 μl, nucleic acid-free water 6.4 μl, DNA template 2 μl .

[0077] The concentrations of upstream primers and downstream primers in the system are both 0.4 pmol / μL.

[0078] Nucleic acid-free water was used as a negative control.

[0079] PCR amplification reaction program: pre-denaturation at 95°C, 5min; 30× (denaturation at 95°C, 30s; annealing at 60°C, 30s; extension at 72°C, 30s); extension at 72°C, 10min.

[0080] The results showed that the pr...

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Abstract

The invention discloses a fluorescent PCR detection method for drug resistance gene mcr (mobile colistin resistance)-4/5/8. A protection primer combination provided by the invention consists of single-stranded DNA represented by sequence 1 to sequence 6 in the sequence table. The SYBR dye-based fluorescent quantitative PCR method for the three mcr genes is established. The detection method has thetechnical advantages of high speed, high sensitivity and strong specificity, and is suitable for monitoring the prevalence of polymyxin drug resistance gene mcr-4/5/8 in clinical and animal husbandry.

Description

technical field [0001] The invention relates to a molecular biology detection method of bacteria in the field of biotechnology, in particular to a detection method of drug resistance gene mcr-4 / 5 / 8 fluorescent quantitative PCR. Background technique [0002] Colistin is considered to be the "last line of defense" in the treatment of multidrug-resistant negative bacterial infections, and its drug resistance rate has remained at a relatively low level for a period of time. Since 2016, the colistin-resistant gene mcr-1, which is mediated by a plasmid and capable of high-frequency horizontal transfer, was reported for the first time in my country. At present, more than 40 countries and regions in the world have successively reported mcr-1 gene. world wide attention. At present, seven new colistin resistance genes mcr-2-8 have been discovered one after another. These eight mcr genes can be transmitted through plasmids, which not only promote the development of clinical colistin r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11C12R1/19
CPCC12Q1/686C12Q1/689C12Q2563/107C12Q2545/114
Inventor 王少林史晓敏李一鸣刘园园
Owner CHINA AGRI UNIV
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