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Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit

A Shigella, Shigella genus technology, applied in the field of microbial detection, can solve the problems of Shigella missed detection, expensive biochips, poor stability, etc., to save time, reduce labor costs and time costs, The effect of avoiding missed detection

Inactive Publication Date: 2013-12-18
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex composition of food and clinical specimens, the extracted nucleic acid may still contain PCR inhibitory components, resulting in false negative results, resulting in missed detection of Shigella
[0007] Biochips can systematically identify Shigella groups and serotypes, but biochip detection requires expensive instruments, poor stability, low reproducibility and other technical difficulties and expensive costs still restrict its further development and application

Method used

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  • Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit
  • Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit
  • Shigella flora/serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Design and synthesis of primers for Freund's type 6 O antigen encoding gene wzx

[0078] Search for 10 full-length sequences of Freund's type 6 O antigen-encoding gene wzx (Freund's type 6) in Genbank, import 10 full-length sequences of wzx (Fundi's type 6) gene into Mega4 software, and use the alignment function for alignment The conservative sequence of wzx (Freund 6) gene was analyzed, and the conservative sequence segment was obtained. The base sequence is shown in SEQ ID No. 14 in the sequence table.

[0079] Input the wzx (Frankenstein type 6) gene sequence into primer premier6 software, and automatically analyze and obtain multiple primer design schemes. On this basis, manually adjust the primer position and sequence length, set the Tm value to 55±5°C, and the GC content of 35%-65%. As far as possible, no hairpin structure and primer dimers are produced, and no error is caused. Perform the Blast analysis on the above-mentioned manually adjusted primer desig...

Embodiment 2

[0083] Example 2 Screening test of Shigella detection primers

[0084] Screening of wzx (Freund 6) gene primer options in Table 2 mainly evaluates the specificity and sensitivity of the primers.

[0085] Specificity evaluation: Extract Escherichia coli ATCC25922, Enterobacter sakazakii, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes, Vibrio cholerae, Bacillus cereus, jejunum Total DNA of Campylobacter, Campylobacter coli, Enterobacter sakazakii, Yersinia enterocolitica, Aeromonas hydrophila, etc. Take 10μL of extract for each bacterium and mix it into a comprehensive DNA template as a specific As a template for verification, the DNA content was determined to be 183.37ng / μL using Nanodrop.

[0086] Dilute the primers into 100μM stock solution with TE (pH8.0) solution, and then prepare the corresponding solution. Configure the reaction system as follows: Taq DNA polymerase 0.4μL (5U / μL), 5×PCR buffer (Tris·HCl100mM (PH8.3), KCl250mM, tween-200.2%) ...

Embodiment 3

[0098] Example 3 Establishment of multiple PCR detection kit for identification of Shigella flora and serotype

[0099] The kit consists of 2× reaction system buffer, DNA polymerase, 10× primer mixture, positive control, and deionized water. The specific components are as follows: 2×PCR Buffer (Tris·HCl40mM (PH8.3), KCl100mM , Tween-200.08%, 0.0006ng / μL pET28a, 1mm dNTP, 8mm MgCl 2 ); 25×DNA polymerase (2U / μL); 10× primer mixture (each primer concentration including IAC primers is 2um), positive control (Freund 2a, Freund 6 and Dysentery I Mixed template with Shigella Sonnei, 10 each 6 CFU / mL).

[0100] The reaction system tested by the kit is 25μL, and the configuration is as follows: 2×PCR Buffer 12.5μL; 25×DNA polymerase 1μL; 10×primer mixture 2.5μL; template 2μL, deionized water 7μL.

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Abstract

The invention discloses a Shigella flora / serotype multiplex-PCR (polymerase chain reaction) detection primer set and kit. The detection primer set is composed of primer pairs for detecting Shigella sonnei, type-1-5 Shigella flexneri, type-I Shigella dysenteriae, type-6 Shigella flexneri, and Shigella. According to the invention, a multiplex-PCR detection method based on a common PCR platform is built; according to the method, multiplex-PCR reaction is carried out on total DNA of bacteria extracted from a sample to be detected in a same reaction system by using the primer pairs obtained through analysis and design, and through electrophoretic analysis on a reaction product, whether the sample is Shigella can be determined and Shigella flora / serotype can be judged.

Description

Technical field [0001] The invention belongs to the field of microbial detection, and relates to a multiple rapid PCR detection primer set and kit for Shigella flora / serotype. Background technique [0002] Shigellosis is a highly contagious and severely harmful gram-negative intestinal pathogen. Shigellosis caused by its clinical infection is one of the most important infectious diseases in developing countries. It is also the main pathogen of diarrhea in developed countries. There are nearly 165 million Shigella dysentery cases worldwide each year, of which 163 million occur in developing countries and cause 1.1 million deaths, of which more than 60% are children under 5 years of age. Due to poor sanitation in the vast rural areas of my country, there are often reports of epidemics of dysentery and diarrhea caused by Shigella contamination of water sources or food. [0003] Shigella belongs to the genus Shigella, divided into four groups: Shigella dysentery (group A), Shigella f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCY02A50/30
Inventor 王晓艳王雷王彦威张志强
Owner 北京卓诚惠生生物科技股份有限公司
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