Diarrheogenic escherichia coli detection kit and detection and typing method thereof

A detection kit, Escherichia coli technology, applied in biochemical equipment and methods, material excitation analysis, microbial determination/inspection, etc., can solve the problems of low accuracy, low detection efficiency, single detection object, etc. Simple, high sensitivity, avoid false positive effect

Active Publication Date: 2013-07-24
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented describes an assay that can detect different types of bacterial pathogens associated with gastrointestinal disorders such as enteritis or inflammatory bowel disease. It uses specialized primer/probe pairs designed specifically against each type of organism involved. By combining these two techniques together it allows quicker identification and more accurate testing without any chance of mistakenly identifying other strains causing symptoms like hemorrhoids or abdominal pain.

Problems solved by technology

The technical problem addressed in this patents relates to improving the speed at diagnosis and analysis of specific types of microorganisms that may have harmful effects on human health or other organism's physiological functions during their natural occurrence. Current diagnostic tools require complex procedures with limited sensitivity and precision due to factors like contamination from previous tests.

Method used

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  • Diarrheogenic escherichia coli detection kit and detection and typing method thereof
  • Diarrheogenic escherichia coli detection kit and detection and typing method thereof
  • Diarrheogenic escherichia coli detection kit and detection and typing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Embodiment 1 Preparation of Diarrheal Escherichia coli Detection Kit

[0113] 1. Synthesize primers and probes for nine genes of stp, sth, lt, aggR, eaeA, escV, stx1, stx2 and ipaH, namely SEQ ID NO: 1-SEQ ID NO: 27, wherein each probe sequence 3' Both ends are connected with DABCYL, and the fluorescent groups corresponding to each probe sequence are as follows:

[0114] SEQ ID NO:3 corresponds to HEX, SEQ ID NO:6 corresponds to FAM, SEQ ID NO:9 corresponds to ROX, SEQ ID NO:12 corresponds to Cy5,

[0115] SEQ ID NO:15 corresponds to Cy5, SEQ ID NO:18 corresponds to HEX, SEQ ID NO:21 corresponds to ROX, SEQ ID NO:24 corresponds to ROX, and SEQ ID NO:27 corresponds to FAM;

[0116] The Homo-tag sequence (SEQ ID NO:28) was synthesized and prepared into a stock solution with a concentration of 50 μM;

[0117] 2. Separately prepare PCR buffer, MgCl 2 solution, dNTP solution, rTaq enzyme solution;

[0118] 3. Take the PCR reaction eight tubes, add PCR buffer, MgCl 2 sol...

Embodiment 2

[0127] 1. Synthesize primers and probes for nine genes of stp, sth, lt, aggR, eaeA, escV, stx1, stx2 and ipaH, namely SEQ ID NO:1-SEQ ID NO:27, wherein each probe sequence 3' Both ends are connected with BHQ, and the fluorescent groups corresponding to each probe sequence are as follows:

[0128] SEQ ID NO:3 corresponds to FAM, SEQ ID NO:6 corresponds to ROX, SEQ ID NO:9 corresponds to Cy5, SEQ ID NO:12 corresponds to HEX,

[0129] SEQ ID NO:15 corresponds to FAM, SEQ ID NO:18 corresponds to FAM, SEQ ID NO:21 corresponds to Cy5, SEQ ID NO:24 corresponds to HEX, and SEQ ID NO:27 corresponds to ROX;

[0130]The Homo-tag sequence (SEQ ID NO:28) was synthesized and prepared into a stock solution with a concentration of 50 μM;

[0131] 2. Separately prepare PCR buffer, MgCl 2 solution, dNTP solution, rTaq enzyme solution;

[0132] 3. Take the PCR reaction eight tubes, add PCR buffer, MgCl 2 solution, dNTP solution, and rTaq enzyme solution, so that they meet the following final...

Embodiment 3

[0140] Example three specificity analysis

[0141] Obtained 204 strains related to Enterobacter, cocci, Vibrio and other bacteria, using the detection kit of Example 1 to carry out specificity experiments and using positive strains as a control, the situation of the detected strains is shown in Table 1, and the detection results were all negative.

[0142] Table 1 Specificity analysis results

[0143]

[0144]

[0145] The results in Table 1 show that the detection kit of Example 1 has good detection specificity, no non-specific amplification occurs, and the occurrence of false positives caused by non-specificity is avoided.

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Abstract

The invention provides a diarrheogenic escherichia coli detection kit and a detection and typing method thereof, which are suitable for the technical field of molecular biology and microbiological detection. The detection kit comprises a PCR (Polymerase Chain Reaction) buffer solution, MgC12, dNTP, DNA (Deoxyribonucleic Acid) polymerase, primers of stp, sth, lt, aggR, eaeA, escV, stx1, stx2 and ipaH genes, probes SEQ ID NO:1-SEQ ID NO:27 and a Homo-tag the sequence of which is SEQ ID NO:28. The detection and typing method comprises the following steps of: obtaining and processing a sample, and adding the processed sample to a detection kit; carrying out fluorescent quantitative PCR reaction; and obtaining a detection result. The detection and genotyping method provided by the invention has the advantages of good specificity, high sensitivity, simple and convenient operation, easiness in identification of the result and is suitable for rapid screening and genotyping of diarrheogenic escherichia coli.

Description

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Claims

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Application Information

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Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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