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Primers for specific gene of Escherichia coli and detection method of Escherichia coli

An Escherichia coli and specific gene technology, applied in the biological field of Escherichia coli, can solve the problems of serious cross-reaction, easy contamination of immunological methods, inability to detect damaged bacteria and dead bacteria, etc., and achieves sensitive and specific test results, convenient and fast detection, Detect quick and cheap effects

Inactive Publication Date: 2019-09-13
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The bacteriological culture method needs to go through enrichment culture, selective separation, observation of morphological characteristics, physiological and biochemical reactions, serological identification and other processes, generally takes 4 to 7 days, the operation is cumbersome, time-consuming and labor-intensive, with low sensitivity and specificity, and Damaged and dead bacteria cannot be detected; immunological methods are prone to contamination, and have serious cross-reactions, many false positives, and low sensitivity; the biggest advantage of nucleic acid probe detection technology is its strong specificity, but there are also problems in probe detection technology. Certain problems, such as the sensitivity is not high enough; to detect a kind of bacteria, it is necessary to prepare a probe

Method used

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  • Primers for specific gene of Escherichia coli and detection method of Escherichia coli
  • Primers for specific gene of Escherichia coli and detection method of Escherichia coli
  • Primers for specific gene of Escherichia coli and detection method of Escherichia coli

Examples

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Effect test

Embodiment 1

[0035] Example 1: Specific target gene screening and primer design

[0036] 1. Local BLAST analysis

[0037] Download the whole genome sequence of Escherichia coli from NCBI (http: / / www.ncbi.nlm.nih.gov / genome / ), perform a local BLAST search on the local nucleic acid database, and obtain the comparison results of each sequence fragment of the strain with the database .

[0038] 2. BLAST reconfirmation

[0039] Using the online BLAST function, 2 strategies were used to confirm their species specificity and species identification availability. One strategy is to exclude the target strain during BLAST, and the results show that those without any similar sequences are the specific genes of the target strain; the second strategy is to search within the target strain when BLAST is used, and the results return a large number of similar sequences to the target strain The consensus sequences of different strains of the species. Combine the two strategies, and finally find out the t...

Embodiment 2

[0042] Embodiment 2: the establishment of detection method

[0043] 1. Extraction of DNA

[0044] (1) Use the bacterial genomic DNA mini-extraction kit of Beijing Zhuangmeng International Biogene Technology Co., Ltd. for extraction and recovery, the steps are as follows:

[0045] ①Take 5mL of bacterial culture solution, centrifuge at 12,000rpm for 1 minute, and aspirate the supernatant as much as possible.

[0046] ② Add 500 μL of cell suspension to the centrifuge tube with the bacterial pellet left, use a pipette or vortex shaker to thoroughly suspend the bacterial cell pellet, and incubate at 37°C for 30 minutes. Mix by inversion several times every 10 minutes. Centrifuge at 12,000rpm for 2 minutes, and try to suck up the supernatant.

[0047] ③Add 225 μL buffer A to the cell pellet and shake until the cell is completely suspended.

[0048] ④Add 6 μL RNaseA solution to the tube, shake for 15 seconds, and place at room temperature for 5 minutes.

[0049] ⑤ Add 10 μL of p...

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Abstract

The invention discloses a method for detecting Escherichia coli on basis of a specific gene of Escherichia coli. The specific gene of Escherichia coli is a hypothetical protein gene which has been registered in Genbank under the registration number 13702648; the specific gene widely exists in Escherichia coli, and has no obvious similarity with other species; and the specific gene has the characteristics of intraspecific commonality and interspecific specificity. Four primers, including a pair of external primers B3 and F3 as well as a pair of internal primers BIP and FIP, are designed on basis of the gene; and then, the primers are applied in loop-mediated isothermal amplification (LAMP), and the external primers B3 and F3 are further applied in polymerase chain reaction (PCR). The nucleotide sequence of the primers is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Identification systems of two kinds of Escherichia coli, namely a PCR reaction system and a LAMP reaction system, are established according to the primers involved; and thus, the identification systems have the characteristics of being good in specificity, high in sensitivity, rapid and reliable indetection result as well as easy to operate. The detection method of Escherichia coli is used for detecting Escherichia coli in water, foods, beverages and cosmetics.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli, and more specifically relates to a method for detecting Escherichia coli based on specific genes of Escherichia coli. Background technique [0002] Escherichia coli (Escherichia coli), also known as Escherichia coli, is classified in the Enterobacteriaceae family and belongs to the genus Escherichia. Most Escherichia coli are normal resident bacteria in the intestinal tract of humans and animals. Escherichia coli is continuously excreted with feces, polluting the surrounding environment, water sources, food, etc. When taking a sample for inspection, the more E. coli in the sample, the more serious the sample is contaminated by feces, and the greater the possibility of the presence of enteric pathogenic bacteria in the sample. Therefore, hygienic bacteriological inspections should be carried out for drinking water, food, and beverages. E. coli infection can be contracted by drinking con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/6844C12Q1/10C12N15/11C12R1/19
CPCC12Q1/689C12Q1/686C12Q1/6844C12Q2565/125C12Q2531/119
Inventor 宋玉竹石耀强李超张阿梅夏雪山韩芹芹张金阳
Owner KUNMING UNIV OF SCI & TECH
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