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Kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof

A technology of pathogenic bacteria and kits, applied in the field of multiplex PCR detection, can solve the problems of less research in the detection of bacterial pathogens, achieve good application prospects, reduce amplification preference, and have the effects of strong specificity

Inactive Publication Date: 2016-03-30
ICDC CHINA CDC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of pathogen detection, this platform has been used to establish the detection of respiratory viruses, enteroviruses, and encephalitis-related arboviruses, but less research has been done on the detection of bacterial pathogens

Method used

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  • Kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof
  • Kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof
  • Kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Design of specific chimeric primers

[0055] By consulting OIE, national standard, industry standard and related literature, determine the pathogenic genes or species-specific genes of 12 common diarrheal pathogens, namely bfpA gene and eaeA gene of E. coli EPEC, elt gene and stp gene of E. coli ETEC , Sth gene, stx1 and stx2 genes of E. coli EHEC, aggR gene of E. coli EAEC, virA gene of E. coli EIEC, virA gene of Shigella, ompC gene of Salmonella, hipO gene of Campylobacter jejuni, Campylobacter coli The ceuE gene of Vibrio cholerae, the ompW gene of Vibrio cholerae, the toxR gene of Vibrio parahaemolyticus, the ail gene of Yersinia enterocolitica. Check the literature and various materials, citing existing or self-designed primer sequences for the above genes. When designing primers, download the relevant nucleic acid sequences from the GenBank database, and use software such as DNAStar and PrimerPremier5.0 to select the conservative regions to design primers....

Embodiment 2

[0066] Example 2 Establishment of multiple PCR detection system for simultaneous detection of 12 diarrhea pathogens

[0067] 1. Preparation of multiplex PCR detection template

[0068] (1) Construction of standard plasmid: Amplify the nucleic acid DNA of the corresponding pathogen with the 3'-end part of the specific chimeric primer (that is, the part after the chimeric primer sequence is removed from the universal primer sequence), and the amplified product is connected to the vector plasmid pMD19-TSimple , Using E. ColiJM109 as the host strain, construct a standard plasmid. A total of 15 standard plasmids were constructed for 15 detection targets.

[0069] (2) Extraction and concentration determination of standard plasmids: QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was used to extract plasmids from host bacterial cultures carrying standard plasmids; the concentration of nucleic acid solution was determined by NanoDropND-1000 with ultra-micro spectrophotometer.

[0070] (3...

Embodiment 3

[0103] Example 3 Validation of multiplex PCR for simultaneous detection of 12 diarrhea pathogens

[0104] 1. Specificity experiment

[0105] 279 strains were used to verify the specificity of the present invention, including 207 strains belonging to the detection scope of the present invention: 43 strains of Vibrio cholerae, 20 strains of Vibrio parahaemolyticus, 6 strains of Campylobacter jejuni, 6 strains of Campylobacter coli, 24 strains Salmonella, 10 strains of Shigella, 5 strains of EIEC, 19 strains of EAEC, 26 strains of EPEC, 45 strains of ETEC, and 2 strains of EHEC; 72 strains not included in the detection scope of the present invention: 40 strains of non-pathogenic E. coli, 18 strains of Aeromonas, 6 strains of Vibrio mimicus, 6 strains of Vibrio fluvialis, 1 strain of Vibrio vulnificus and 1 strain of Edwards tarda. For Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli, Salmonella, EIEC&Shigella and EAEC, only one electrophoretic band w...

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Abstract

The invention provides a kit capable of simultaneously detecting 12 diarrhea pathogenic bacteria and application thereof, and belongs to the technical field of multiplex PCR detection. The kit comprises specific chimeric primers corresponding to the 12 common diarrhea pathogenic bacteria and a pair of internal reference primers, and the nucleotide sequences of the specific chimeric primer pairs are shown in SEQ ID NO.1-32 respectively. According to the kit, reaction conditions are optimized by arranging a multiplex PCR system, and accurate detection on the 12 diarrhea pathogenic bacteria is achieved by analyzing the length of a product through single-tube one-time PCR and agarose electrophoresis. The kit has the advantages of being accurate in detection, high in sensitivity, strong in specificity, simple, convenient and rapid and suitable for preliminary screening of common pathogenic bacteria in clinical diarrhea samples and epidemiological investigation of diarrhea, and a good application prospect is achieved.

Description

Technical field [0001] The present invention relates to the field of multiplex PCR detection technology, in particular to a method for simultaneously detecting 10 diarrhea pathogens, a detection kit and applications thereof. Background technique [0002] Infectious diarrheal disease is a common disease with a high incidence, especially in children, causing a huge burden of disease. About 10%-40% of infectious diarrhea are caused by bacterial pathogens, including pathogenic Escherichia coli, Shigella, Salmonella, Vibrio and Campylobacter. Bacterial isolation and culture is the gold standard for infection laboratory diagnosis, but the operation is cumbersome, time-consuming, and the positive rate is low, and the experience of the operator has a greater impact on the results. Nucleic acid detection is currently playing an increasingly important role in pathogen detection, especially PCR-based technology, which has received extensive attention due to its advantages of simple operati...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2600/166C12Q2537/143C12Q2565/125C12Q2545/101Y02A50/30
Inventor 张京云管红霞凌霞阚飙
Owner ICDC CHINA CDC
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