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Triple real-time fluorescent PCR method and kit for detecting three streptococci at the same time

A real-time fluorescence, streptococcus technology, used in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc.

Inactive Publication Date: 2014-11-19
海南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no report of a kit for simultaneous detection of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae

Method used

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  • Triple real-time fluorescent PCR method and kit for detecting three streptococci at the same time
  • Triple real-time fluorescent PCR method and kit for detecting three streptococci at the same time
  • Triple real-time fluorescent PCR method and kit for detecting three streptococci at the same time

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Preparation of Three Streptococcus DNA Templates

[0084] Three kinds of streptococcus [Streptococcus agalactiae (ATCC 13813), Streptococcus dysgalactiae (ATCC 35666), Streptococcus iniae (ATCC 29178) were tested according to the instructions of TIANamp Bacteria DNA Kit, and the three kinds of streptococci were obtained from the American Type Culture Collection ( ATCC)] Genomic DNA extraction and purification, add the corresponding reagents in order:

[0085] (1) Take 1.5 mL of bacterial liquid, centrifuge at 10,000 r / min for 1 min, discard the supernatant, add 200 μL of buffer GA, and suspend the bacterial cells thoroughly;

[0086] (2) Add 20 μL proteinase K (20mg / mL), and add 220 μL buffer solution GB, mix thoroughly, and act in a 70°C water bath for 10 minutes;

[0087] (3) Add 220 μL of absolute ethanol, mix thoroughly for 15 seconds, transfer the resulting solution (including flocculent precipitate) to the adsorption column CB3 after brief centrifugation, centrif...

Embodiment 2

[0094] Preparation of positive control substance

[0095] 1. Design and synthesis of PCR primers

[0096] Select the cpsF gene of Streptococcus agalactiae as the target gene, the MIG gene of Streptococcus dysgalactiae as the target gene, and the 16SrRNA gene of Streptococcus iniae as the target gene, and after BLAST analysis, design and synthesize primer pairs, as shown in Table 1:

[0097] Table 1 Three PCR primer pairs for Streptococcus

[0098]

[0099] 2. Utilize the target gene amplification primers SA-F / SA-R, SD-F / SD-R, SI-F / SI-R of the above-mentioned three kinds of streptococcus, respectively amplify Streptococcus agalactiae by PCR method cpsF gene, Streptococcus dysgalactiae MIG gene and Streptococcus iniae 16S rRNA gene. The reaction system of the three-segment gene PCR is as follows:

[0100] 10×PCR Buffer 2.5μL dNTP (2.5mM for each) 2.0 μL upstream primer 1.0 μL downstream primer 1.0 μL Taq DNA Polymerase (5U / μL) 0...

Embodiment 3

[0115] Design and synthesis of primers and probes for triple real-time fluorescent PCR of three kinds of streptococci

[0116] According to the genome DNA sequences of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae released in Genbank, combined with bioinformatics methods, the cpsF gene of Streptococcus agalactiae, the MIG gene of Streptococcus dysgalactiae and Streptococcus iniae The conserved region sequence of the 16S rRNA gene was used as the detection target sequence, and 3 pairs of primers and 3 probes were designed and synthesized for the qualitative detection of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae. As shown in table 2.

[0117] Table 2 Triple real-time fluorescent PCR primers and probes for three kinds of streptococci

[0118]

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Abstract

The invention provides a triple real-time fluorescent PCR method and a kit for detecting three streptococci at the same time. The kit comprises: a streptococcus agalactiae detection primer pair which is represented as the Seq ID No.1 and the Seq IN No.2 in the sequence table, a probe which is represented as the Seq ID No.3 in the sequence table, a streptococcus dysgalactiae detection primer pair which is represented as the Seq ID No.4 and the Seq ID No.5 in the sequence table, a probe which is represented as the Seq ID No.6 in the sequence table, a streptococcus iniae detection primer pair which is represented as the Seq ID No7 and the Seq ID No.8 in the sequence table, a probe which is represented as the Seq ID No.9 in the sequence table, three positive comparison sample which are represented as the Seq ID No.16, the Seq ID No.17 and the Seq ID No.18 in the sequence table, a negative comparison sample (ddH2O), a PCR buffering liquid required in fluorescent real-time quantification PCR amplification, dNTP and TaqDNAPolymerase. The method is simple and quick and is good in specificity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a triple real-time fluorescent PCR method and a kit for simultaneously detecting three kinds of streptococci. Background technique [0002] Streptococcus is a genus with a huge population. At least 60 species and subspecies have been found in this genus. There are not many types of pathogenic Streptococcus pathogenic bacteria in aquatic animals. From farmed fish The streptococci that have been isolated on the Internet include Streptococcus iniae (S.iniae), Streptococcus difficile (S. diffucilis), Streptococcus agalactiae (S. agalactiae), Streptococcus mieri (S. milleri), paramammary chain S. parauberis and S. dysgalactiae, but the reported pathogenic streptococci are mainly Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus iniae, which can cause a variety of sea and freshwater fish Streptococcus disease occurs in all major fish farming countries in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
CPCC12Q1/6806C12Q1/6851C12Q2531/113C12Q2537/143C12Q2561/101C12Q2545/113
Inventor 李丹丹徐义刚高慎阳蔡波
Owner 海南出入境检验检疫局检验检疫技术中心
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