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Method for detecting digestive tract pathogens

A pathogen and digestive tract technology, applied in the field of pathogenic microorganism molecular biology, can solve the problems of difficult clinical promotion, complicated operation, low degree of automation, etc., and achieve the effects of simple reagent consumables, stable reaction system, and less environmental pollution.

Active Publication Date: 2015-02-18
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is not highly automated, and the operation is relatively complicated, so it is difficult to promote it clinically.

Method used

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  • Method for detecting digestive tract pathogens
  • Method for detecting digestive tract pathogens
  • Method for detecting digestive tract pathogens

Examples

Experimental program
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Effect test

Embodiment 1

[0056] In the present embodiment, a method for detecting two kinds of pathogenic plasmids (Listeria monocytogenes, Enterovirus 71) is provided, comprising the steps of:

[0057] (1) Obtain the amplified product of the target sequence of the pathogen plasmid to be tested by PCR amplification. The amplification primer set used is shown in Table 1 (the corresponding primer combination in the reaction system 1 or 2 where the pathogen to be detected is located), wherein each primer needs to be prepared with a 10-base tag sequence (acgttggatg) at its 5' end ), the purpose is to increase the molecular weight of the primer sequence to exclude its influence in subsequent steps.

[0058] The reaction system used in PCR amplification is as follows, and all reagents are purchased from Sequenom Company:

[0059]

[0060]The PCR reaction conditions were: 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and a tota...

Embodiment 2

[0079] In this embodiment, a detection method for a clinically collected pathogen mixed infection sample is provided, including the following steps:

[0080] (1) Carry out preliminary treatment to the sample collected clinically, use the kit (QIAGEN company) to extract pathogen DNA and RNA, utilize the kit purchased from Takara company The RT reagent Kit reverse-transcribes the extracted RNA sample into cDNA.

[0081] Steps (2)-(6) are the same as steps (1)-(5) in Example 1, except that the template DNA used in the PCR amplification reaction system is the clinical sample DNA prepared in the above step (1) and cDNA, and the amplification primers and extension primers used are the amplification primers and extension primers corresponding to reaction system 2.

[0082] The following combination image 3 , to analyze and explain the test results of the above-mentioned pathogen mixed infection samples.

[0083] image 3 It is the mass spectrometry detection peak diagram when u...

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Abstract

The present invention provides a method for detecting common digestive tract pathogens. The present invention also provides amplification primers and extension primers for detecting common digestive tract pathogens, and kits comprising these primers.

Description

technical field [0001] The invention relates to the field of molecular biology of pathogenic microorganisms, in particular to a detection method for digestive tract pathogens. The present invention also relates to amplification primers and extension primers for detecting digestive tract pathogens, and kits comprising these primers. Background technique [0002] Infectious diseases caused by pathogens in the digestive tract are clinically common and frequently-occurring diseases. Gastrointestinal infectious diseases usually refer to gastrointestinal diseases caused by pathogens such as bacteria and viruses, which can occur all year round, and are more common in summer and autumn. The common ones are cholera, typhoid, viral hepatitis, and bacillary dysentery and other infectious diarrhea. Pathogens of digestive tract infection often cause water and food pollution, which can cause outbreaks of infectious diseases and emergencies such as collective disease, directly endangerin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70
CPCC12Q1/6883C12Q2600/16C12Q1/689Y02A50/30
Inventor 姜永强陈唯军杨银辉刘利成管彦芳朴静子
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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