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Primers and method for detecting tehA gene in Escherichia coli

A technology for detecting Escherichia coli and primers, which is applied in the field of molecular biology, can solve the problems of complicated sequencing technology steps, low sensitivity, long time consumption, etc., and achieves the effects of preventing strains from continuing to spread, high resolution, and high detection rate.

Inactive Publication Date: 2012-07-04
上海中优医药高科技股份有限公司
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Problems solved by technology

Gel electrophoresis is low cost and easy to operate, but it has low sensitivity and is prone to false negatives; sequencing is the gold standard for detecting gene mutations, but sequencing technology steps are cumbersome, time-consuming, and prone to contamination

Method used

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  • Primers and method for detecting tehA gene in Escherichia coli
  • Primers and method for detecting tehA gene in Escherichia coli
  • Primers and method for detecting tehA gene in Escherichia coli

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Embodiment

[0022] 1. Prepare a set of primers for detecting tehA gene carrying in Escherichia coli by solid-phase phosphoramidite triester method, including:

[0023] TehA gene forward primer: 5′-tgagtttgtttcctgcaacg-3′;

[0024] TehA gene reverse primer: 5'-agcttcttcagggtgagatce-3'.

[0025] 2. Detection method:

[0026] (1) Take the clinical sample isolates identified as positive for Escherichia coli after culture, inactivate them at high temperature, and extract the sample DNA with a commercial bacterial genomic DNA magnetic bead extraction kit (Shenzhen Yirui Biotechnology Co., Ltd., YP03002), Dilute to 30ul (concentration 10ng / ul), store at -20°C, and wait for use; the standard Escherichia coli strain (preservation unit: China Agricultural Microorganism Culture Collection Management Center, preservation number: Number: 25922), high-temperature inactivation, extract sample DNA with a commercial bacterial genomic DNA magnetic bead extraction kit (Shenzhen Yirui Biotechnology Co., L...

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Abstract

The invention discloses primers and a method for detecting tehA gene in Escherichia coli. The primers for detecting the tehA gene in the Escherichia coli are characterized by comprising a tehA gene forward primer 5'-tgagtttgtttcctgcaacg-3' and a tehA gene reverse primer 5'-agcttcttcagggtgagatcc-3'. The detection method comprises the following steps of: taking a clinical sample isolate which is cultured and identified as Escherichia coli positive, deactivating at high temperature, and extracting sample DNA serving as a detection sample; taking an Escherichia coli strain which does not carry the tehA gene, deactivating at high temperature, and extracting sample DNA serving as a negative control; artificially synthesizing a tehA sequence, and constructing a recombinant plasmid, which carries the tehA gene, serving as a positive control; and respectively performing polymerase chain reaction (PCR). The invention has the advantages of high detection rate and high repeatability.

Description

technical field [0001] The invention relates to a method for detecting the carrying condition of a disinfectant-resistant tehA gene in Escherichia coli (Escherichia coli, ECO), and belongs to the technical field of molecular biology. Background technique [0002] Bacterial efflux pump systems can be divided into five categories: ATP-binding cassette transporter family (ATP-binding cassette family, ABC), major facilitator superfamily (MFS), drug and metabolite transporter superfamily (the much larger drug / metabolite transporter superfamily, DMT) and the related small multidrug resistance family (SMR), the multidrug and toxic compound extrusion family (MATE ), tolerance-nodulation-division family (the resistance-nodulation-division family, RND). ABC are energy pumps and the rest are proton pumps. Proton pump is a secondary drug (compound) transport system mediated by the proton motive force (PMF) produced by bacteria through proton coupling exchange. Bacteria express resist...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/19
Inventor 傅咏南张奕王校
Owner 上海中优医药高科技股份有限公司
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