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Plasmid fragment carrying novel gene qepA3

A plasmid and fragment technology, applied in genetic engineering, plant genetic improvement, microbial-based methods, etc., can solve the problems of low prevalence, transmission and infection, and achieve the effect of preventing explosive epidemics and reducing use

Inactive Publication Date: 2015-11-11
王冬国
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Korean researchers disclosed that the prevalence of the qepA gene in South Korea is very low, Chinese researchers found that the qepA gene is commonly found in food-borne animals such as pigs, chickens, and ducks that carry Enterobacteriaceae. In this way, the potential spread of the qepA gene and Infection and even outbreaks are very likely, so the detection and monitoring of qnr, aac(6')-Ib-cr, qepA and oqxAB genes has an urgent practical need and significance

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  • Plasmid fragment carrying novel gene qepA3
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  • Plasmid fragment carrying novel gene qepA3

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1 Identification of the plasmid fragment carrying the new gene of qepA3

[0026] 1. Isolation and identification of strain EC3157

[0027] 1.1 Materials

[0028] Bacterial susceptibility card: AST-GN13 from bioMerieux, France. AST-GN13 drug-sensitive types include: amikacin, ampicillin, ampicillin / sulbactam, aztreonam, cefazolin, cefepime, cefotetan, ceftazidime, ceftriaxone, ciproxa Star, ertapenem, gentamicin, imipenem, levofloxacin, nitrofurantoin, piperacillin / tazobactam, tobramycin.

[0029] 1.2 Method

[0030] Apparatus identification: transfer positive bacterial strains from ICU patients of Taizhou Municipal Hospital to blood plates for isolation and culture (at 35°C containing 5% CO 2 Cultivate in the incubator for 16-18h), and then carry out bacterial identification and drug susceptibility test on the VITEK2 machine according to the operating procedures for the isolated pure bacteria.

[0031] Supplementary drug susceptibility test: smear 0.5 Mc...

Embodiment 2

[0051] Example 2 Plasmid transduction experiments to study the function of plasmid fragments carrying the new qepA3 gene

[0052] 1. Method

[0053] (1) The donor strain is EC3157 strain, and the recipient strain is E.coliJ53Az R (resistant to sodium azide). The donor bacteria and recipient bacteria were inoculated on LB plates, respectively, and cultured overnight at 35°C. Pick a single colony and inoculate them in 4 mL of LB broth, and culture at 37°C and 220 r / min until the logarithmic growth phase. Take 0.5 mL of donor and recipient bacteria in 4 mL of LB broth, and culture overnight at 37°C. Zygotes were screened on trypan soy agar (TSA) plates containing sodium azide (300 mg / L) and amifloxacin (0.03 mg / L). Incubate at 35°C for 18-24h. Extract the plasmid of the drug-resistant bacterial strain (the steps are the same as in Example 1), and carry out the detection of the plasmid carrying the qepA3 new gene by the PCR method.

[0054] The primers and sequencing primers...

Embodiment 3

[0065] Example 3 Gene recombination experiment to study the function of the plasmid fragment carrying the qepA3 new gene

[0066] 1. Method

[0067] (1) The PCR product in Example 2 (the nucleotide sequence is SEQIDNO.1) is connected to the pMD19-T vector, and the connection product is added to 100 μl of E.coliJM109 in a competent state for transformation, and the PCR product containing IPTG, X-Gal, Amp Cultivate on a plate, and sequence the plasmids extracted from IPTG, X-Gal, and Amp drug-resistant strains (the steps are the same as in Example 1), and detect whether the plasmid fragment carrying the new qepA3 gene is inserted into the genome. Then clone this segment of sequence into the pET32a vector. The insertion site of this segment of sequence is at the DraI and BamHI restriction sites on the pET32a vector. It is prepared according to conventional methods to obtain the new genes carrying qepA3, rmtB and bla respectively. CTX-M-14 Gene pET32a recombination plasmid. Tran...

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Abstract

The invention discloses a plasmid fragment carrying novel gene qepA3. Sequences of the plasmid fragment are shown in SEQ ID NO.1. The plasmid fragment is found in a genome of Escherichia coli EC3157. Discovery of the plasmid fragment, on the one hand, is of important theoretical and practical significance to genetic level study on molecular mechanisms of resistance transfer in bacteria; on the other hand, has certain guidance on clinical medication and is helpful for clinical reduction in usage of certain antibacterial agents, reduction in selective pressure of resistance gene transfer of this agents, and prevention of explosive epidemic of drug-resistant bacteria.

Description

technical field [0001] The invention belongs to the field of molecular biology technology and drug-resistant bacteria monitoring, and relates to a newly discovered plasmid closely related to drug resistance of Escherichia coli. Background technique [0002] Escherichia coli is one of the most common pathogenic bacteria in medicine and veterinary clinics, which has caused serious damage to human health and the development of animal husbandry. Antibacterial drugs have played an important role in controlling Escherichia coli infection, but with the widespread use of antibacterial drugs, drug-resistant strains have emerged rapidly, and multi-drug resistance is very common, especially the multi-drug resistance caused by plasmid-mediated Escherichia coli has become a Serious problems that threaten global medicine and public health. [0003] According to literature reports, the mechanisms of quinolone resistance mediated by plasmids include: qnr gene factors, aminoglycoside acetyl...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12N1/21C12N15/10C12N15/87C12R1/19
Inventor 王冬国陶国仙阮桂英陈佳玉
Owner 王冬国
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