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LAMP primer group, kit and detection method for rapid test of bacterial polymyxin drug resistance gene mcr-1

A polymyxin and drug-resistant gene technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of cumbersome operation process and long time consumption, and achieve high sensitivity and specificity Good, highly specific effect

Inactive Publication Date: 2017-06-13
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] for mcr-1 Gene detection, traditional microbial culture method and drug sensitivity test method can provide good clinical value, but due to various reasons such as time-consuming, cumbersome operation process, expensive equipment and other reasons, it has not been widely used in clinical practice. Promote the use, thus developing a simple and rapid bacterial polymyxin resistance gene mcr-1 The detection method has great application prospects

Method used

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  • LAMP primer group, kit and detection method for rapid test of bacterial polymyxin drug resistance gene mcr-1
  • LAMP primer group, kit and detection method for rapid test of bacterial polymyxin drug resistance gene mcr-1
  • LAMP primer group, kit and detection method for rapid test of bacterial polymyxin drug resistance gene mcr-1

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Experimental program
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Embodiment 1

[0039] Embodiment 1, rapid detection bacterial polymyxin resistance gene mcr-1 The establishment of the kit

[0040] Rapid detection of bacterial polymyxin resistance genes mcr-1 The kit includes the following components: (1) LAMP detection primer set; (2) DNA polymerase; (3) LAMP reaction solution; (4) color reagent; (5) positive control and negative control.

[0041] (1) LAMP detection primer set

[0042] bacterial polymyxin resistance gene mcr-1 (GenBank accession number: KX886345.1), for its 6 regions, LAMP primer design was carried out, after a large number of primer specificity experiments, a LAMP detection primer set with good specificity was selected, including a pair of inner primers and a pair of outer primers. The nucleotide sequences of the primer and a pair of loop primers are as follows:

[0043] Internal primer 1: 5'- GCGATGGGATAGGTTTGGCTGTGTTGCCGTTTTCTTGAC-3' (SEQ ID NO: 1);

[0044] Internal primer 2: 5'-TGCTGACGATCGCTGTCGGCACATAGCGATACGATGAT-3' (SEQ ID NO:...

Embodiment 2

[0054] Embodiment 2, rapid detection bacterial polymyxin resistance gene mcr-1 Methods

[0055] Rapid detection of bacterial polymyxin resistance gene using the kit of Example 1 mcr-1 ,Specific steps are as follows:

[0056] (1) Extract the DNA of the sample to be tested:

[0057] Bacterial genomic DNA was extracted by boiling method.

[0058] (2) Use the LAMP detection primer set to perform isothermal gene amplification reaction on the DNA of the sample to be tested:

[0059] The 25 μL system of isothermal gene amplification reaction is as follows: the final concentration of inner primers 1 and 2 is 8 pmol / µL, the final concentration of outer primers 1 and 2 is 1 pmol / µL, and the final concentration of loop primers 1 and 2 is 4 pmol / µL. µL, 12.5 µL of reaction solution, 8 U of DNA polymerase, 50 ng of DNA of the sample to be tested, make up to 25 µL with sterilized deionized water, and then add 20 µL of sealing solution;

[0060] The isothermal gene amplification program...

Embodiment 3

[0064] Embodiment 3, sensitivity experiment

[0065] Take the positive control substance (that is, the constructed drug containing bacterial polymyxin resistance gene mcr-1 Fragment pMD19-T plasmid), measure its concentration and calculate the copy number, dilute according to 10-fold concentration gradient, select 1.0×10 0 ~1.0×10 9 The concentration of copies / μL was used as a sample for experimentation; the detection method in Example 2 and the conventional PCR method were used for detection respectively.

[0066] The primer sequences used in conventional PCR detection methods are as follows:

[0067] MCR-1 -F:CGGTCAGTCCGTTTGTTC (SEQ ID NO: 7);

[0068] MCR-1 -R: CTTGGTCGGTCTGTAGGG (SEQ ID NO: 8).

[0069] Adopt the detection result of embodiment 2 detection method as figure 2 Shown: its pair mcr-1 The minimum detection limit of the gene is: 1.0×10 4 copies / μL.

[0070] The detection results by conventional PCR method are as follows: image 3 Shown: its pair mc...

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Abstract

The invention discloses a LAMP primer group, a kit and a detection method for rapid test of a bacterial polymyxin drug resistance gene mcr-1. The kit comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. The six specific primers and a DNA polymerase with strand displacement activity are adopted for amplification of a DNA sample at 60-65 DEG C, and color changes in a reaction tube can be observed to judge whether amplification occurs or not after addition of a color developing agent. The LAMP primer group, the kit and the detection method have advantages of quickness, high efficiency, simplicity and convenience in operation, high specificity, high sensitivity, simplicity and convenience in detection, suitableness for onsite detection and the like and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the detection of pathogenic microorganisms, in particular to a rapid detection of bacterial polymyxin resistance genes mcr-1 The LAMP primer set, kit and detection method. Background technique [0002] Polymyxin belongs to the family of cationic polypeptide antibiotics, including A, B, C, D, and E. The antibacterial spectrum is similar to each other and has a wide range. It is generally considered to be the last line of defense against multidrug-resistant Gram-negative bacterial infections. Clinically commonly used are polymyxin B (polymyxin B) and polymyxin E (colistin, colistin). [0003] In recent years, a new gene was discovered mcr-1, Can make bacteria highly resistant to polymyxins, which are widely present in Enterobacteriaceae, including strains with epidemic potential, from pigs and patients in southern China. A known, mcr-1 The gene product belongs to the phosphoethanolamine transferase family, which can catalyze...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/689
Inventor 田国宝黄曦钟兰兰曾昆姣沈聪
Owner SUN YAT SEN UNIV
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