Diarrhea pathogenic bacteria multi-gene detection system and its kit and application

A technology for gene detection and pathogenic bacteria, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problem that the detection and diagnosis methods of diarrhea pathogenic bacteria cannot meet clinical needs and cannot accurately identify multiple pathogenic bacteria. , the inability to diagnose the basis of pathogenic bacteria and other problems, to achieve the effect of low-cost pathogenic diagnosis, low cost and good convenience

Active Publication Date: 2017-08-22
HUADONG HOSPITAL +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current conventional detection methods have the disadvantages of low detection rate, long time-consuming, especially the inability to accurately identify multiple pathogenic bacteria at the same time, and cannot provide timely, comprehensive and accurate pathogen diagnosis basis for clinical practice, leading to the widespread use of broad-spectrum Empirical treatment with antimicrobial drugs leads to problems such as low curative effect, failure to control diarrhea in time, high incidence of drug-resistant strains, and disturbance of digestive flora
[0009] In summary, the current detection and diagnosis methods for pathogenic bacteria in diarrhea cannot meet the clinical needs, and the development of new technologies is urgently needed

Method used

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  • Diarrhea pathogenic bacteria multi-gene detection system and its kit and application
  • Diarrhea pathogenic bacteria multi-gene detection system and its kit and application
  • Diarrhea pathogenic bacteria multi-gene detection system and its kit and application

Examples

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Embodiment 1

[0051] 1. The composition of the kit

[0052] The diarrhea pathogen multiple gene detection kit of the present embodiment detection kit comprises: primer mixture, PCR buffer (5×PCR Buffer), MgCl 2 solution, dNTPs, hot-start DNA polymerase and reverse transcriptase mix, UDG enzyme, positive and negative controls.

[0053] PCR buffer, hot-start DNA polymerase and reverse transcriptase mix were all from Qiagen (Catalog No.: 210212).

[0054] The hot-start DNA polymerase and reverse transcriptase are mixed together to form a mixed solution, wherein the concentration of the hot-start DNA polymerase in the reaction system is 0.1U / μL, and the concentration of the reverse transcriptase in the mixed solution is 0.1U / μL.

[0055] The positive control solution is a plasmid mix that includes all gene targets of interest.

[0056] The negative control solution was nuclease-free ultrapure water.

[0057] The primer mixture includes: the nucleotide sequence of the forward primer and the n...

Embodiment 2

[0107] The rest of the diarrhea pathogenic bacteria multiple gene detection kit of the present embodiment is the same as that of Example 1, except that the primer mixture only includes: respectively for Campylobacter jejuni, Shigella, Clostridium difficile, Salmonella enteritidis , Salmonella typhimurium, Enterotoxigenic Escherichia coli, Enterohaemorrhagic Escherichia coli, Enteropathogenic Escherichia coli, Enteroadhesive Escherichia coli, Enteroinvasive Escherichia coli, Escherichia coli Forward and reverse PCR amplification primers for detection of bacteria O157, Vibrio, and Yersinia enterocolitica, and forward and reverse PCR amplification primers for detection of human DNA internal reference and system quality control internal reference. It is used for the simultaneous detection of 13 common pathogenic bacteria in infectious diarrhea.

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Abstract

The invention relates to a diarrhea pathogenic bacteria multi-gene detection system and its kit and application. The detection system has 15 pairs of primers, wherein 13 pairs of diarrhea pathogens primers, one pair of human genome beta-actin primers and one pair of system quality controlled beta-actin primers are included. Diarrhea pathogens refer to campylobacter jejuni, shigella, clostridium difficile, salmonella enteritidis, salmonella typhimurium, enterotoxigenic escherichia coli, escherichia coli O157, vibriones, yersinia enterocolitica and the like. The diarrhea pathogenic bacteria multi-gene detection system and its kit do not require routine culture and other steps, synchronous detection and analysis of the various diarrhea pathogens can be directly conducted on a stool sample in the same reaction system, the shortcomings that a routine detection method is low in flux and low in detection rate and takes much time are made up for, a comprehensive, accurate and low-cost pathogen diagnosis is provided for clinical use for the first time, and an important reference is provided for individualized medication and accurate medical treatment.

Description

technical field [0001] The invention relates to a multiple gene detection product and a detection system used in the product, belonging to the field of biotechnology. Background technique [0002] Infectious diarrhea is an intestinal infectious disease caused by a variety of pathogenic bacteria, and is one of the most common digestive system infectious diseases worldwide. There are about 2 billion diarrheal diseases in the world every year, and about 800 million people suffer from diarrhea every year in my country. Infectious diarrhea has become the second largest infectious disease affecting human health after respiratory tract infection. [0003] Etiological diagnosis is of great significance to the prevention and treatment of infectious diarrhea. "World Organization of Gastroenterology Global Guidelines - Acute Diarrhea in Adults and Children: Global Perspectives (2012)" and "Expert Consensus on the Diagnosis and Treatment of Acute Infectious Diarrhea in Chinese Adults ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10
CPCC12Q1/689C12Q2600/16C12Q2600/166Y02A50/30
Inventor 赵虎张艳梅李敏李冬杨长青陈敏杨丽华保志军吴勇陈洁
Owner HUADONG HOSPITAL
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