PCR detection method for streptococcus equi subsp zooepidemicus and kit used thereby

A Streptococcus equi and detection reagent technology, applied in the field of epidemiology and health detection, can solve the problem of low homology, achieve high sensitivity, shorten the detection time, and facilitate the operation

Inactive Publication Date: 2009-12-09
范红结 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both Streptococcus equi subsp. zooepidemicus and Streptococcus suis type 2 express fibronectin-binding protein, but the homology between them is less than 20%, while other Streptococcus species do not express the protein

Method used

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  • PCR detection method for streptococcus equi subsp zooepidemicus and kit used thereby
  • PCR detection method for streptococcus equi subsp zooepidemicus and kit used thereby
  • PCR detection method for streptococcus equi subsp zooepidemicus and kit used thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: establishment and optimization of multiplex PCR reaction system

[0036] 1.1 Design and synthesis of primers

[0037] According to the public sequences AY263781, DQ363208, and AY938342 of the M protein gene, fibronectin binding protein, and superoxide dismutase A gene of the subspecies of zooepidemicus, three pairs of primers were designed by using DNAStar software, among which primers P1 and P2 amplified Streptococcus equi zooepidemicus The length of the subspecies M protein gene fragment is 750bp, and the length of the fibronectin-binding protein gene fragment of Streptococcus equi subsp. zooepidemicus amplified by primers P3 and P4 is 540bp. The length of the dismutase A gene fragment is 230bp. The three pairs of primers were all synthesized by TaKaRa Company, and the sequences of the three pairs of primers are:

[0038] M-like protein

[0039] Sense primer P1: 5'CTA TCG GTG GTC GTA ATG GAG A 3'

[0040] Antisense primer P2: 5'ACC TGC CAT AAC TGC AA...

Embodiment 2

[0095] Example 2: Multiplex PCR specificity test

[0096] 2.1 Strains

[0097] 2.1.1 Positive strains

[0098] Streptococcus equi zooepidemic subspecies ATCC35246, isolated from Sichuan, my country in 1977, has ATCC collection

[0099] CVCC552 strain of Streptococcus equi subsp. zooepidemicus was purchased from China Culture Collection Center.

[0100] CVCC555 strain of Streptococcus equi subsp. zooepidemicus was purchased from China Culture Collection Center.

[0101] CC strain of Streptococcus equi subspecies zooepidemicus, a gift from Jiangsu Academy of Agricultural Sciences.

[0102] CT strain of Streptococcus equi subspecies zooepidemicus, kindly donated by Jiangsu Academy of Agricultural Sciences.

[0103] CY strain of Streptococcus equi subspecies zooepidemicus, a gift from Jiangsu Academy of Agricultural Sciences.

[0104] 2.1.2 Negative strains

[0105] Streptococcus equi subspecies CVCC1892 was purchased from China Culture Collection Center.

[0106] Streptoco...

Embodiment 3

[0119] Embodiment 3: the sensitivity test of multiple PCR detection method

[0120] Using Streptococcus equi subsp. zooepidemicus ATCC35246 strain, genomic DNA was extracted by proteinase K cleavage method as amplification template.

[0121] Use a spectrophotometer to measure the concentration of genomic DNA, start from 100ng / ml 10-fold dilution, and use different dilutions of bacterial genomic DNA as a template for multiplex PCR amplification. The multiplex PCR reaction system and reaction conditions described in 1.7 were adopted, and the method was the same as that described in Example 1. The effects of different template concentrations on multiplex PCR amplification effects were compared.

[0122] The results showed that the target fragment could be amplified when the template amount reached 10 ng / ml, and the amplified target band gradually increased with the increase of the DNA template amount.

[0123] It can be seen from this that the multiplex PCR amplification method...

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Abstract

The invention belongs to the field of epidemiology and sanitation tests, and relates to a PCR quick detection kit for quickly detecting streptococcus equi subsp zooepidemicus and a PCR quick detection method. Particularly, the kit of the invention comprises three pairs of primers which are aimed at M-like proteins (SzP), fibronectin binding proteins (Fnz), superoxide dismutase A (SodA) coding gene respectively and specifically, so the kit can detect the three virulence factors of the streptococcus equi subsp zooepidemicus at the same time. The multiplex PCR detection method is quick and simple, can directly detect pathologic materials, avoids a normal complex process of bacteria isolation, cultivation and biochemical identification, reduces detection time and simplifies operation to allow for skillful use after simple training. The kit is high in sensitivity and can detect micro pathogen. And the kit is high in specificity and can distinguish streptococcus equi subsp zooepidemicus from other kinds of streptococcus without any cross reaction.

Description

technical field [0001] The invention belongs to the field of epidemiology and sanitation detection. Specifically, the present invention relates to a PCR rapid detection kit for rapid detection of Streptococcus equi subsp. zooepidemicus and a detection method using the kit. Background technique [0002] Streptococcus suis is an important bacterial infectious disease that seriously endangers the pig industry in the world and can cause human infection. The main pathogens of the disease are Streptococcus equi subsp. zooepidemicus and Streptococcus suis, type 2. At present, the etiological diagnosis of Streptococcus suis mainly depends on the isolation and cultivation of pathogenic bacteria, which is time-consuming and labor-intensive. [0003] Streptococcus equi subspecies zooepidemicus is the main pathogen of streptococcus suis in my country, and there is still a lack of rapid and sensitive detection methods. Therefore, the development of rapid and sensitive new detection ki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/46
Inventor 范红结李芳陆承平
Owner 范红结
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