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Lamp primers and detection methods for the detection of Shigella flexneri serotypes 2a and xv

A technology for detecting Shigella flexneri and serotypes, applied in the field of molecular biology detection, can solve problems such as unfavorable promotion and use, and achieve the effects of high specificity, good specificity and high sensitivity

Active Publication Date: 2022-07-19
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current fluorescent quantitative PCR system requires multiple reactions to be performed at the same time to detect serotypes, and a fluorescent quantitative PCR instrument is required, which is not conducive to the promotion and use at the grassroots level

Method used

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  • Lamp primers and detection methods for the detection of Shigella flexneri serotypes 2a and xv
  • Lamp primers and detection methods for the detection of Shigella flexneri serotypes 2a and xv
  • Lamp primers and detection methods for the detection of Shigella flexneri serotypes 2a and xv

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 DNA template preparation

[0065] DNA templates were prepared using the boiled method. Take a single colony on a Shigella plate that has been cultured overnight, put it into an eppendof tube containing 100 μL of sterile water, and shake to mix. Boil in water at 100°C for 10 minutes, place on ice for 5 minutes, centrifuge at 13,000 rpm for 10 minutes, and take the supernatant as a template.

Embodiment 2

[0066] Example 2 LAMP primer design

[0067] Primer design for wzx, gtrII, gtrX and opt genes was performed using Primer Explorer V5 software (https: / / primerexplorer.jp / v5_manual / index.html). LAMP primers include a pair of inner primers (FIP and BIP), and a pair of outer primers (F3 and B3). A pair or one loop primer (LF and / or LB) can be added to the reaction system to increase the specificity and speed of the LAMP reaction. The specificity of primers was tested in NCBI database by BLAST method.

[0068] Table 1 is the information of the designed LAMP primers. See the position of the primers on the gene. figure 1 .

Embodiment 3

[0069] Example 3 LAMP reaction process

[0070] LAMP reactions were performed using WarmStart Colorimetric LAMP 2X Master Master Mix (New England Biolabs Inc., USA). The LAMP reaction system included 25 μL of primer mix (40 pmol each for FIP and BIP primers, 10 pmol each for F3 and B3 primers, and 20 pmol each for LF and / or LB primers), 12.5 μL master mix and 1 μL DNA template. Incubate the mixed LAMP reaction tube at 61°C for 20 minutes. The color change was observed with the naked eye, and the positive reaction LAMP master mix changed from red to yellow. The negative reaction LAMP master mix remains red.

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Abstract

The present invention provides a LAMP primer and a detection method for detecting Shigella flexneri 2a and Xv serotypes. The present invention designs LAMP primers according to the type-specific antigenic determinants and group-specific antigenic determinants of the main serotypes 2a and Xv of Shigella flexneri, wherein the 2a serotype is (gtrII and wzx genes are positive, gtrX and opt genes are negative) ), Xv serotype (gtrX, opt and wzx genes positive, gtrII gene negative). The method does not require expensive equipment and instruments, and can rapidly, accurately and specifically detect Shigella flexneri 2a and Xv serotypes.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a LAMP primer and a detection method for detecting Shigella flexneri 2a and Xv serotypes. Background technique [0002] According to statistics, the main prevalent serotypes of Shigella flexneri in my country are 2a and Xv serotypes. According to the statistical data from 1991 to 2000, the main prevalent Shigella in my country is Shigella flexneri, accounting for 86%. Among them, the dominant serotype is 2a, accounting for 80%. The Xv serotype first appeared in Henan in 2000, and then replaced the 2a serotype as the dominant serotype from 2002 to 2006. In 2007, among Shigella isolated from Gansu and Anhui provinces, Xv serotype was also the dominant serotype, accounting for 67% and 54% of the isolates, respectively. In a surveillance data from 2003 to 2013 based on sentinel hospitals across the country, among the 2912 Shigella isolates, 1610 were Shigella flexneri, of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/125C12Q2565/113
Inventor 金东李沙
Owner ICDC CHINA CDC
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