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Nucleic acid multiplex PCR detection kit for 11 intestinal pathogenic bacteria and its application

A technology for detection kits and pathogenic bacteria, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, microorganisms, etc., can solve problems such as interactions, achieve high detection sensitivity, reduce detection costs, increase throughput and The effect of detection sensitivity

Active Publication Date: 2018-12-11
NANJING MOKOBIO BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The technical problem solved by the present invention: In order to solve the problem of interaction between multiple pairs of primers in the same reaction system, the present invention adopts the multiplex PCR amplification technology of general primers

Method used

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  • Nucleic acid multiplex PCR detection kit for 11 intestinal pathogenic bacteria and its application
  • Nucleic acid multiplex PCR detection kit for 11 intestinal pathogenic bacteria and its application
  • Nucleic acid multiplex PCR detection kit for 11 intestinal pathogenic bacteria and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 111

[0049] Example 1 Nucleic acid multiplex PCR detection kit for 11 kinds of intestinal pathogenic bacteria

[0050] The kit consists of primer mix A, primer mix B, PCR reaction buffer, enzyme system, positive control, negative control and DEPC water.

[0051] PCR reaction solution A is a mixture of primer mixture A and PCR reaction buffer; PCR reaction solution B is a mixture of primer mixture B and PCR reaction buffer.

Embodiment 2

[0052] Operation and result determination of embodiment 2 kit

[0053] (1) Extraction of bacterial genomic DNA

[0054] Pick the feces the size of a grain of rice (take the mucus, pus and blood as much as possible), put them in a centrifuge tube with 0.5ml of normal saline, shake and mix well, and centrifuge at 13,000rpm for 2min, remove the supernatant, and precipitate for later use. Use the fecal nucleic acid extraction kit (product number: DP328) from Tiangen Biochemical Technology Co., Ltd. to extract nucleic acid in the sample processing area according to the instructions.

[0055] (2) Preparation of amplification system

[0056] The following experiments were carried out using the kit of Example 1. After the components of the kit were completely dissolved at room temperature, they were quickly shaken and mixed. 25 μL PCR amplification system A reaction system is: 18 μL each of PCR reaction solution A, 2 μL enzyme system, 2-5 μL template (including nucleic acid extracte...

Embodiment 3

[0065] Embodiment 3 kit specificity experiment

[0066] Select 8 enterovirus samples including coxsackievirus, poliovirus, echovirus, enteroadenovirus, norovirus, astrovirus, rotavirus group A, and zaravirus, as well as campylobacter jejuni and mononuclear virus 4 kinds of intestinal bacteria of cell proliferative Listeria, Vibrio alginolyticus, and Aeromonas, using the kit described in Example 1 to operate and determine the results according to the method described in Example 2, the detection results of the above 12 pathogens No characteristic peaks with peak sizes shown in Table 2 were shown, indicating that the kit of the present invention has good specificity.

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Abstract

The invention discloses a multiple PCR detection kit for 11 intestinal pathogen nucleic acid and application of the detection kit. The detection kit comprises primers for amplifying 11 intestinal pathogens which include vibrio cholerae serotype O1, vibrio cholerae serotype O139, salmonella, Shigella, vibrio parahaemolyticus, Yersinia enterocolitica, enterophathogenic escherichia coli (EPEC), enteroinvasive escherichia coli (EIEC), enteroaggregative escherichia coli (EAEC), enterotoxigenic escherichia coli (ETEC) and escherichia coli O157:H7. The multiple PCR detection kit has the advantages that the kit is capable of performing multiple detection, high in sensitivity and fast and convenient to use; specific primer sequences are used to guarantee detecting result reliability; the detection method is simple to operate, time saving, labor saving, high in detection throughput, low in reagent consumable cost, capable of directly detecting the nucleic acid extracted from encephalitis pathogens, low in detection platform and staff technical level requirements and capable of being widely popularized in conventional detection.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a kit for detecting nucleic acids of 11 kinds of intestinal pathogenic bacteria by using a multiplex PCR amplification method mediated by universal primers and an application thereof. Background technique [0002] Diarrhea is a common and frequently-occurring disease in summer and autumn, with serious harm, complex causes, and a wide range of susceptible populations. The main pathogens include bacteria, viruses, and biological factors, and bacterial infection is the most common cause of diarrhea. In addition, the national food safety standard (GB29921-2013) stipulates the limit requirements for pathogenic bacteria in food. Therefore, intestinal pathogenic bacteria are important detection items for clinical diagnosis and food safety inspection. [0003] Enteropathogenic bacteria mainly include diarrhea-causing Escherichia coli, Vibrio cholerae, Salmonella, Shigella, Yersinia enterocol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12Q1/04C12R1/63C12R1/42C12R1/19C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125Y02A50/30
Inventor 刘文干谢国祥金鑫丁洁朱坤张平
Owner NANJING MOKOBIO BIOTECH
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