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Method for detecting bacterium serotypes

A technology of serotype and bacteria, applied in the direction of biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc., can solve problems such as high cost and complexity

Active Publication Date: 2020-05-05
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when performing multiplex real-time PCR that needs to distinguish each target sequence, these improved real-time PCR methods need to use double or even triple the number of probes, which is more complicated and costly than traditional real-time PCR methods

Method used

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  • Method for detecting bacterium serotypes
  • Method for detecting bacterium serotypes
  • Method for detecting bacterium serotypes

Examples

Experimental program
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Effect test

Embodiment 1O157

[0312] The detection of embodiment 1.O157 serotype Escherichia coli

[0313] In this example, the reagents described in Table 1 (9 detection probes, 38 mediator probes, 38 upstream primers, 38 downstream primers, and 1 universal primer) and the detection protocol described in Table 2 were used , to detect samples containing Escherichia coli O157 serotype.

[0314] In short, in this example, a 25 μL PCR reaction system was used for real-time PCR, and the PCR reaction system included: 1×PCR buffer (67mM Tris-HCl, 50mM KCl and 0.085mg / mL BSA), 6.0mM MgCl 2 , 0.2 mM dNTPs, 2.0 U of polymerase TaqHS (Takara), various reagents described in Table 1 (used at the indicated working concentrations), and 5 μL of E. coli DNA of serotype O157. The reaction conditions of real-time PCR were: 95°C, 5min; then 50 cycles (95°C, 20s and 63°C, 1min). After PCR is finished, carry out melting curve analysis according to following program: 95 ℃, 2min; The temperature was raised from 40°C to 95°C, ...

Embodiment 2 38

[0316] Embodiment 2. Thirty-eight multiple detections

[0317] In this example, the reagents described in Table 1 (9 detection probes, 38 mediator probes, 38 upstream primers, 38 downstream primers, and 1 universal primer) and the detection protocol described in Table 2 were used , to contain 37 kinds of bacterial serotypes (respectively from O29 type E. coli, O29 type E. coli, O26 type E. coli, O169 type E. coli, O161 type E. , E. coli O1, E. coli O157, E. coli O148, E. coli O27, E. coli O167, E. coli O6, E. coli Gp6, E. coli O15, E. coli O159, E. coli Gp12 , E. coli O25, E. coli O166, E. coli O91, E. coli Gp2, E. coli O86, E. coli O111, E. coli O121, E. coli O145, E. coli O55, E. coli Gp9 , E. coli O63, E. coli O18, E. coli O128, E. coli O119, E. coli O153, E. coli O103, E. coli O113, E. coli O104, E. coli O45, E. coli O142 DNA) and control DNA (Escherichia coli yccT gene) samples were tested.

[0318] In short, in this example, a 25 μL PCR reaction system was used for re...

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Abstract

The application provides a method for detecting bacterium serotypes. Through the adoption of the method, the existence of various bacterium serotypes (such as escherichia coli O serotypes, escherichiacoli H serotypes, escherichia coli K serotypes, salmonella O serotypes, salmonella H serotypes, vibrio parahaemolyticus O serotypes, vibrio parahaemolyticus K serotypes, shigella O serotypes and vibrio cholerae O serotypes) in samples can be detected at the same time. In addition, the application further provides a probe set and a reagent kit comprising one or more probe sets. The probe sets andthe reagent kit can be used for implementing the method disclosed by the invention. In addition, the application further provides the reagent kit. The existence or the level of a plurality of bacterium serotypes in the samples can be detected at the same time in a round of reactions.

Description

technical field [0001] The present application relates to multiplex detection of nucleic acid molecules. In particular, the application provides a method for detecting bacterial serotypes, which can simultaneously detect multiple (such as 2, 5, 10, 15, 20, 25, 30 or more) bacteria of serotypes in a sample existence in. In addition, the present application also provides a probe set, and a kit comprising one or more of the probe sets, and the probe set and the kit can be used to implement the method of the present invention. In addition, the present application also provides a kit, which can simultaneously detect multiple (such as 2, 5, 10, 15, 20, 25, 30 or more) serotypes of bacteria in a sample in one round of reaction The presence. Background technique [0002] Bacterial serotyping refers to the further classification of bacteria of the same species based on the differences in bacterial antigenic determinants. Bacterial serotyping is a common international and domestic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6844C12Q1/689C12Q1/10C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2533/101C12Q2527/107C12Q2521/319Y02A50/30
Inventor 廖逸群李庆阁杜琛许晔
Owner XIAMEN UNIV
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