Glycopeptide antibiotic drug-resistance gene detection kit

A glycopeptide antibiotic and detection kit technology, applied in the field of molecular biology, can solve the problems of difficult clinical treatment of vancomycin-resistant Enterococcus infection, threat of anti-infective treatment, and increased glycopeptide resistance rate, etc. Broad market potential for clinical applications, easy-to-read results, and the effect of reducing false positives

Pending Publication Date: 2017-05-10
重庆威斯腾生物医药科技有限责任公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

Glycopeptide antibiotic resistance genes can be carried by transposons and plasmids, and transmitted through conjugation between bacteria, so that the rate of glycopeptide resistance in enterococci continues to increase, and many vancomycin-resistant enterococci are also resistant to other antibiotics (such as β-lactam and aminoglycoside antibiotics) drug resistance, causing great difficulties in the clinical treatment of vancomycin-resistant enterococcal infections
On the other hand, some methicillin-resistant Staphylococcus aureus clinical isolates have been found to be attenuated in susceptibility to vancomycin and teicoplanin, and transferable vanB resistance has been found in clinical isolates of Streptococcus bovis The determinant, the vanA gene cluster was found in the clinical isolates of Bacillus circulans, indicating that the glycopeptide resistance gene is not only transmitted among enterococci, but also can be transmitted to other bacteria other than enterococci, which brings great benefits to the future anti-infective treatment It is of great significance to the development and development of efficient and accurate detection of glycopeptide resistance genes in clinical adjuvant therapy

Method used

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  • Glycopeptide antibiotic drug-resistance gene detection kit
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  • Glycopeptide antibiotic drug-resistance gene detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Target Fragment Primer and Probe Design

[0022] Find the gene sequences VanA, VanB, VanC, VanH, VanR, VanS, and VanX of the glycopeptide antibiotic resistance genes in the NCBI database, and design 3 probes for each drug resistance gene using chip probe design software (ArrayDesigner4.2) In addition, a probe of the 16sRNA gene was designed as a positive quality control probe (PC) and a randomly synthesized probe was used as a negative quality control probe (NC). The corresponding probe sequences are shown in SEQ ID NO.1~23.

[0023] The NCBI online design software (Primer-BLAST) was used to design the PCR amplification primers of VanA, VanB, VanC, VanH, VanR, VanS and VanX genes, and simultaneously design the amplification primers of 16sRNA gene. In order to make the chip results more intuitive, the 5' end of the reverse amplification primer (R) was labeled with a CY3 fluorescent dye, and the corresponding primer sequences are shown in SEQ ID NO.24~39.

Embodiment 2

[0024] Example 2 Preparation of chip

[0025] The parallel probes in Example 1 were spotted on the amino-modified glass substrate according to the order of Table 1 to obtain a gene chip containing probes (such as figure 1 shown). The probe concentration was 30 μM, 0.2 μL per spot, and then incubated at 80°C for 1.5 h.

[0026]

Embodiment 3

[0027] Embodiment 3 detection method

[0028] 1. Genome extraction of samples to be tested

[0029] Using the bacterial genome DNA extraction kit, follow the steps in the operating instructions to extract the genome of the sample to be tested as the amplification template.

[0030] 2. PCR amplification of the target fragment

[0031] After a lot of experimentation, the target gene PCR amplification system and PCR reaction procedure for Van detection were determined. The primers for VanA, VanB, VanC, VanH, VanR, VanS, VanX and 16SrRNA amplification of each Van gene were prepared respectively for PCR amplification buffer system, and the reagents contained in the PCR amplification system with a total volume of 20 μL were: Premix LATaq 10 μL, F primer (10 μM) 1 μL, F primer (10 μM) 1 μL, amplification template 2 μL, double distilled water 6 μL.

[0032] The PCR reaction program was: 95°C for 5 min; 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[003...

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Abstract

The invention discloses a glycopeptide antibiotic drug-resistance gene detection kit. The glycopeptide antibiotic drug-resistance gene detection kit comprises probes 1 to 23, and corresponding sequences are shown as SEQ ID NO. 1 to SEQ ID NO. 23; the invention further discloses a method for detecting a glycopeptide antibiotic drug-resistance gene by utilizing the kit; when the glycopeptide antibiotic drug-resistance gene detection kit disclosed by the invention is used for detecting, the credibility of an experiment result needs to be evaluated at first; the detection result of the group is credible only when detection results of positive and negative quality control contrasts are positive (+) and negative (-). When the results displayed by the three probes of a detected object are consistent, whether a sample is infected by the glycopeptide antibiotic drug-resistance gene or not can be judged; when the results displayed by the three probes of the detected object are inconsistent, whether the sample to be detected is positive or negative cannot be judged, and the detection kit needs to be used for redetecting or existing other detection methods are used for further identification. The glycopeptide antibiotic drug-resistance gene detection kit is simple to operate, rapid and efficient and high in accuracy and the results are easy to read.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a parallel probe, a gene chip, a kit and a detection method for detecting glycopeptide antibiotic resistance genes. Background technique [0002] In the past few decades, antibiotics have been widely used in the treatment of various infectious diseases, but it is precisely because of the widespread use of antibiotics that new forms of antibiotic resistance continue to emerge. Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa are resistant to more than 100 antibiotics used clinically, and have become important bacterial killers that threaten human life. Glycopeptide antibiotics (G1ycopeptides, Dalbahepides) are D-alanyl-D-alanine binding and a class of antibiotics with a heptapeptide structure. , Enterococcus (including Enterococcus faecalis and Enterococcus faecium), Corynebacterium, anaerobic coccus and Listeria monocytogenes, almost all Gram-positive ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6837C12Q2565/501C12Q2563/107
Inventor 周勇申友锋
Owner 重庆威斯腾生物医药科技有限责任公司
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