A kind of detection method of Streptococcus pneumoniae serotype

A Streptococcus pneumoniae detection method technology, applied in the field of serotype detection of Streptococcus pneumoniae, can solve the problems of high price, pollution, and increased nucleic acid aerosol

Active Publication Date: 2015-08-12
江西南兴医疗科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional MLPA technology needs to be opened many times during the operation, which increases the risk of nucleic acid aerosol contamination, and the result detection requires an expensive capillary electrophoresis sequencer, which hinders its application in clinical testing

Method used

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  • A kind of detection method of Streptococcus pneumoniae serotype
  • A kind of detection method of Streptococcus pneumoniae serotype
  • A kind of detection method of Streptococcus pneumoniae serotype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Simultaneous detection of Streptococcus pneumoniae samples mixed with multiple subtypes

[0134] The method can simultaneously detect multiple subtypes contained in a sample, and distinguish them through melting curves with different Tm values. The template is mixed purified 4, 6, 19F Streptococcus pneumoniae genomic DNA, each DNA is 2.5ng.

[0135] The reaction conditions are:

[0136] PCR pre-amplification:

[0137] reaction system

Volume (total volume 50 μl)

concentration

10×PCR Buffer (Mg 2+ Plus)

5μl

1×

dNTP Mixture (2.5mM each)

4μl

0.2mM

Template (bacterial genomic DNA)

2.5ng

0.05ng / μl

Primer 1 (PF1-PF11)

1μl

0.2μM each

Primer 2 (PR1-PR11)

1μl

0.4μM each

TaKaRa Taq (5U / μl)

0.25μl

1U

[0138] Sterile distilled water

Up to 50μl

[0139] Reaction procedure:

[0140] ① 94℃ for 5 minutes

[0141] ② 94℃ 30s

[014...

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Abstract

The invention provides a method for carrying out serotyping on streptococcus pneumoniae by using MLPA (multiplex ligation dependent probe amplification). The method comprises the following steps of: firstly, extracting a sample genome DNA of streptococcus pneumoniae to be detected; carrying out pre-amplification on the extracted genome DNA by using a PCR pre-amplification primer; carrying out hybridization on pre-amplification products of the genome DNA by using an MLPA probe; adding a corresponding fluorescence detecting probe in a reaction system; connecting hybridized MLPA probes by using ligase, and carrying out PCR amplification on the hybridized and connected MLPA probes by using an MLPA universal amplification primer; and analyzing PCR products by using a multicolor fluorescence dissolution curve, so that the serotyping on streptococcus pneumoniae can be realized. The detecting method disclosed by the invention is good in specificity and high in sensitivity; according to the method, 10 kinds of serotypes can be subjected to genotyping simultaneously just in 2-4 hours, so that multiple uncapping operations in traditional MLPA detection are avoided, and the pollution possibility is reduced, therefore, the method can meet high-throughput sample detection, and is especially applicable to inspection departments.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection technology for serotype typing of Streptococcus pneumoniae, and specifically relates to a detection method for serotype typing of Streptococcus pneumoniae, which is suitable for qualitative detection of the serotype of Streptococcus pneumoniae. Background technique [0002] Streptococcus pneumoniae (Streptococcus pneumonia), also known as Diplococcus pneumoniae or pneumococcus, is positive for Gram staining and generally arranged in a double spherical shape under a microscope; it has a capsule structure, and the capsule is the main virulence factor of Streptococcus pneumoniae. According to the structure and composition of capsular polysaccharides, Streptococcus pneumoniae can be divided into 46 serogroups and more than 90 different serotypes. About 20 serotypes / groups are associated with common pneumococcal invasive disease, and most serotypes are harmless to humans. At pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/46
Inventor 黄劭
Owner 江西南兴医疗科技有限公司
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