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Preparation method and application of VRE/MRSA/KPC/NDM-1 detection gene chip

A technology for detecting gene chips and gene chips, applied in the field of gene chip detection, can solve the problems of long experimental period, cumbersome operation, strong experience dependence, etc., and achieve the effects of high specificity, high throughput and good specificity

Inactive Publication Date: 2017-09-05
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional phenotypic detection method has a long experimental cycle, cumbersome operation, and strong experience dependence, which cannot meet the needs of clinical treatment.

Method used

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  • Preparation method and application of VRE/MRSA/KPC/NDM-1 detection gene chip
  • Preparation method and application of VRE/MRSA/KPC/NDM-1 detection gene chip
  • Preparation method and application of VRE/MRSA/KPC/NDM-1 detection gene chip

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Development of VRE / MRSA / KPC / NDM-1 detection gene chip.

[0039] 1. Primer probe design and screening.

[0040] Firstly, the common super-resistant gene sequences were downloaded from the NCBI gene database. After the sequences were downloaded, the ALignX program in the Vector NTI Advance 10 (invitrogen) software package was used to make a global comparison of the drug-resistant gene sequences according to the default parameter settings. Design specific oligonucleotide probes, general and specific primers at the conserved positions of the gene sequence according to the comparison results. After screening, a total of 8 pairs of primers were finally determined, and the reverse primer was bio-labeled at the 5' end as the primer used for the chip; 8 specific detection probes were determined, and the 3' end NH 2 grooming.

[0041] 2. Preparation of oligonucleotide chip and probe array.

[0042] After completing the probe screening, the final probe array was dete...

Embodiment 2

[0050] Example 2: Specificity evaluation of VRE / MRSA / KPC / NDM-1 detection gene chip.

[0051] Specificity is the most important assessment index of a diagnostic method. The gene chip of the present invention uses optimized systems and conditions to detect Escherichia coli, Salmonella, Helicobacter pylori, Vibrio parahaemolyticus, pyogenes, Haemophilus influenzae, attached by Figure 4 It can be seen that the above bacterial detection results were all negative, with good specificity. The present invention detects 5 different kinds of super-drug-resistant genes and 3 related specific genes of pathogenic bacteria. image 3 It can be seen that each gene can be clearly distinguished, indicating that the specificity of the present invention is good.

Embodiment 4

[0052] Example 4: Sensitivity evaluation of VRE / MRSA / KPC / NDM-1 detection gene chip.

[0053] Five different kinds of super drug-resistant genes and related three kinds of pathogen-specific gene plasmid bacteria were used as detection reference products, from 10 5 copies / μL serially diluted to 10 1 copies / μL, for chip detection, the results are shown in the appendix Figure 5 . attached by Figure 5 As can be seen, the detection sensitivity of the chip of the present invention is 3 × 10 3 copies / response.

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Abstract

The invention relates to a preparation method and application of a VRE / MRSA / KPC / NDM-1 detection gene chip. The preparation method includes: preparing specific primers, preparing specific probes, preparing an oligonucleotide chip, and building a multiple PCR system and a hybrid system. By the prepared gene chip, 5 drug-resistant genes and 3 related pathogenic bacteria can be detected at the same time, and the 5 drug-resistant genes and 3 related pathogenic bacteria include Vancomycin-resistant Enterococcus VRE drug-resistant genes VanA and VanB, an encoding New Delhi metallo-beta-lactamase-1 NDM-1 drug-resistant gene, a methicillin-resistant staphylococcus aureus MRSA drug-resistant gene mecA, an encoding carbapenemase KPC gene, an enterococcus faecalis specific gene Fddl, an enterococcus faecium specific gene Sddl and a staphylococcus aureus specific gene nuc. The chip which provides a new detection manner for the confirmation of the drug-resistant genes of common antibiotics resistance phenotypes has advantages of quickness, accuracy, high flux and the like.

Description

technical field [0001] The invention relates to the preparation and application of a VRE / MRSA / KPC / NDM-1 detection gene chip, and belongs to the technical field of gene chip detection. Background technique [0002] Infectious diseases have always been a great threat to human health and life. The discovery of antibiotics in the 1940s made significant progress in the treatment of some infectious diseases. However, with the irrational use and abuse of antibiotics, bacterial resistance Drug rates keep rising. Bacterial drug resistance has developed into a major global public health threat. Infections caused by drug-resistant bacteria make treatment ineffective, increasing patient suffering, loss of labor and even death. Under the pressure of antibiotic selection, some pathogenic bacteria are resistant to antimicrobial drugs, which reduces the efficacy of antimicrobial drugs or even fails them. The trend of drug resistance has also developed from single drug resistance to multip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6837C12Q1/689
Inventor 王升启吴冰刘琪琦陈苏红宋燚
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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