PCR detection kit and detection method for Streptococcus equi

A technology of streptococcus equine and a detection kit, which is applied in the field of microbial biotechnology, can solve the problems of extended detection cycle, unsatisfactory detection, and low specificity, and achieve the effects of fast detection speed, simple operation, and high sensitivity

Inactive Publication Date: 2014-02-05
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are very few domestic studies on the diagnosis of equine gland disease. The traditional methods of isolation and culture, biochemical identification, and serotyping are still widely used in the detection of this bacteria, which is time-consuming and labor-intensive, and is not suitable for the detection needs in production.
There are also studies that use 16SrDNA technology as an auxiliary diagnosis. This diagnostic technology still needs to be verified by sequencing and sequence analysis, and the specificity is not strong, which prolongs the detection cycle (Chen Xinhua, Kang Lichao, Huang Xin, etc., Isolation and Identification of Streptococcus equine epidemic , Chinese Journal of Veterinary Medicine, 2013 1(49): 38-39), which cannot meet the needs of rapid, simple and accurate diagnostic methods for the disease in current clinical and production practices

Method used

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  • PCR detection kit and detection method for Streptococcus equi
  • PCR detection kit and detection method for Streptococcus equi
  • PCR detection kit and detection method for Streptococcus equi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Extraction of the genome of the tested bacteria and PCR amplification of the target gene.

[0032] Genome Extraction

[0033] The purified strain was inoculated in 3 mL of TH liquid medium and cultured overnight. The specific method is as follows:

[0034] (1) Take 1.5mL of bacterial solution cultured overnight, centrifuge, discard the culture medium, then use sterilized deionized water, 400uL spin sedimentation, mix thoroughly, then centrifuge again, 12000rpm, 5min. Repeat the above operation once.

[0035] (2) After discarding the supernatant, add 400uL of TE buffer, mix well, add 100uL (20mg / mL) lysozyme and 5uL RNase, mix well again, and bathe in water at 37°C for 1h30min.

[0036] (3) Add 80 uL of 10% SDS solution, mix thoroughly, and bathe in water at 37°C for 1h30min.

[0037] (4) Add 100uL 5mol / L Nacl solution and 80uL CTAB / Nacl solution, mix well, bathe in 37°C water for 30min, and place at -20°C for 20min.

[0038] (5) Add 500uL phenol: chlorof...

Embodiment 2

[0047] Example 2: Sensitivity detection of equine Streptococcus adenoepidemicus PCR detection kit.

[0048] Such as figure 2Take the overnight culture of Streptococcus equine as shown, count it with a hemocytometer, and divide the bacterial solution by 10 6 , 10 5 , 10 4 , 10 3 , 10-fold dilution, extract DNA for PCR reaction. The analysis of the detection results showed that the minimum detection limit of the established Streptococcus equine epidemics PCR detection kit was 80cfu.

Embodiment 3

[0049] Example 3: Detection of the specificity of the established PCR detection kit for Streptococcus equine.

[0050] Select Staphylococcus aureus, Salmonella, Escherichia coli, and Streptococcus zooepidemicus to detect the specificity of the PCR detection kit for Streptococcus equine. From image 3 It can be seen that only the DNA of Streptococcus equi standard bacteria has the same size as the target fragment. This detection method can be used to specifically detect Streptococcus equi in horse herds.

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Abstract

The present invention relates to a PCR detection kit for detecting Streptococcus equi (further called Streptococcus equi subsp equi). The detection kit comprises primers, dNTP, a buffer and DNA polymerase, wherein the upstream primer is 5'-CTATTAAAGTCTCCATTG-3', and the downstream primer is 5'-AATGTTGTTCAAGCAAATTC-3'. The PCR detection method comprises: providing a sample template requiring detection; adding dNTP, a 10*buffer, primers, DNA polymerase, a sample requiring detection and ddH2O to a PCR thin-walled tube, and uniformly mixing; amplifying the mixture in the thin-walled tube through a PCR instrument; carrying out electrophoresis on the amplification product in electrophoresis equipment; and analyzing and judging the result. According to the present invention, the PCR kit preparation method is simple and is suitable for industrial production; the PCR detection method has characteristics of high Streptococcus equi detection sensitivity, short detection period, rapidness, and strong operability, wherein detection accuracy can achieve template DNA of 80 bacterial while a detection cost is relatively low; and the detection kit and the detection method have important application values in clinical diagnosis of the disease.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology, and the specific content relates to a PCR detection kit which can be used to detect Streptococcus equi (also known as Streptococcus equi subspecies equi), comprising primers, dNTP, buffer and DNA polymerase, The upstream primer is 5'-CTATTAAAGTCTCCATTG-3', and the downstream primer is 5'-AATGTTGTTCAAGCAAAATTC-3'; PCR detection method: provide the sample template to be tested; add dNTP, 10×buffer, primers, DNA to the PCR thin wall tube Polymerase, test sample and ddH 2 O, mix well; make the mixture in the thin-walled tube amplify on the PCR instrument; electrophoresis the amplified product in the electrophoresis device; analyze and judge the result. technical background [0002] Equine Streptococcus (Streptococcus. Suppurative lymphangitis of the head. Therefore, research on S. equine is urgently needed. The United States, Britain and other countries have always attached great importance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12R1/46
CPCC12Q1/14C12Q1/686C12Q2531/113
Inventor 苏艳刘云涛王彩蝶张宝江
Owner XINJIANG AGRI UNIV
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