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Tigecycline resistance gene family tetX multiplex PCR detection kit

A technology for detecting kits and drug-resistant genes, which is applied in the field of molecular biology to achieve the effects of convenient operation, low energy consumption, and high accuracy

Active Publication Date: 2019-09-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can only detect one resistance gene. In the face of the emergence of more and more other genes of the tetX gene family, multiple reactions are required to conduct a more comprehensive detection of related resistance genes.

Method used

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  • Tigecycline resistance gene family tetX multiplex PCR detection kit
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  • Tigecycline resistance gene family tetX multiplex PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A tigecycline-resistant gene family tetX multiplex PCR rapid detection kit, including rapid PCR mixed solution (2×Rapid Taq Master Mix), ultrapure water, primer probe mixed solution, standard substance (tetX1, tetX2, tetX3 , tetX4, tetX5), negative control substance. The rapid PCR mixture (2×Rapid Taq Master Mix) contains Taq DNA Polymerase, elongation promoting factor, dNTP and optimized buffer system. The primer-probe mixture includes primers tetX1-F, tetX1-R, tetX2-F, tetX2-R, tetX3-F, tetX3-R, tetX4-F, tetX4-R, tetX5-F, tetX5-R.

[0027] The sequence of the tetX1 primer pair is shown in SEQ No.1 and SEQ No.2, the sequence of the tetX2 primer pair is shown in SEQ No.3 and SEQ No.4, and the sequence of the tetX3 primer pair is shown in SEQ No.5 and SEQ No. 6, the sequences of the tetX4 primer pair are shown in SEQ No.7 and SEQ No.8, and the sequences of the tetX5 primer pair are shown in SEQ No.9 and SEQ No.10.

[0028] The above-mentioned tetX1, tetX2, tetX3, tetX4, ...

Embodiment 2

[0031] 2.1 Materials and methods

[0032] The using method of kit of the present invention: all should set up positive control and negative control in each sample detection, and standard result is as follows: figure 1 . The bacteria solution to be tested was directly used as a template for multiplex PCR detection.

[0033] The multiplex PCR reaction system (total volume 50 μL) is: primer-probe mixture: 10 μL (working concentration: 0.2 μM); template: 2 μL of bacteria solution to be tested; rapid PCR mixture (2×Rapid Taq Master Mix): 25 μL; ultrapure Water: 13 μL.

[0034] Multiplex PCR reaction program: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 59°C for 15s, extension at 72°C for 20s, a total of 30 cycles; final extension at 72°C for 5min.

[0035] The multiplex PCR products were detected by electrophoresis on 2.0% agarose gel with a voltage of 110V and a working time of 120min.

[0036] 2.2 Detection and identification of wild-type stra...

Embodiment 3

[0041] 3.1 Primer design:

[0042] According to the reported literature, the DNA sequences of tetX1, tetX2, tetX3, tetX4, and tetX5 were obtained from NCBI as the target genes. Primer Premier 5.0 software was used to design primers with different PCR product lengths on different target genes to ensure that different drug resistance genes could be clearly distinguished in the end. Then use the Vector NTI software to compare and analyze the specificity of the designed primers and the homology with the target gene, so that the homology of each primer with the target gene is 100%, and the homology with other genes is kept low. The origin ensures that the primers are universal within the target gene and specific between genes, and finally determine the sequence of 5 pairs of identification primers. Pattern diagram such as image 3 shown.

[0043] 3.2 Specific detection between primers

[0044] see Figure 4 , using a single pair of primers to perform single-fold PCR amplificat...

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Abstract

The invention provides a tigecycline resistance gene family tetX multiplex PCR detection kit. The tigecycline resistance gene family tetX multiplex PCR detection kit consists of a rapid PCR mixed solution, ultrapure water, a primer probe mixed solution, a standard substance and a negative reference substance, wherein the primer probe mixed solution comprises primer pairs of tetX1, tetX2, tetX3, tetX4, tetX5 and the like. According to the invention, a sensitive and efficient PCR detection method is established by using multiplex PCR test means, and strains containing tetX1, tetX2, tetX3, tetX4 and tetX5 genes can be rapidly and effectively detected. The tigecycline resistance gene family tetX multiplex PCR detection kit has the advantages of low cost, high efficiency, convenience in operation, high accuracy, less energy consumption, less environmental pollution and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a novel tigecycline drug-resistant gene family tetX multiple PCR detection kit. Background technique [0002] Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics, which have good antibacterial effects on Gram-positive and negative bacteria, chlamydia, mycoplasma and Plasmodium falciparum, and are widely used in the prevention of bacterial infections in humans and livestock And treatment, and because low-dose tetracycline can promote the growth of animals, it is also widely used in agriculture and aquaculture. The extensive use and discharge of tetracycline antibiotics has promoted the development of tetracycline-resistant bacteria and resistance genes (tetracycline resistance genes, TRGs). There are 44 TRGs reported so far, which are mainly divided into three categories according to the mechanism of action: efflux pump genes, ribosome protection genes and enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2600/16C12Q2537/143C12Q2565/125C12Q2527/125
Inventor 冯友军徐勇昌季凯蒋承建
Owner ZHEJIANG UNIV
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