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Fluorescent quantitative PCR method for detecting toxin-producing streptococcus suis and corresponding kit

A Streptococcus suis, fluorescence quantitative technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low sensitivity and time-consuming, achieve high sensitivity, wide application range, improve The effect of efficiency

Pending Publication Date: 2020-08-14
GUANGZHOU MICROCHIP BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, a lot of research has been done on Streptococcus suis at home and abroad, and many effective detection methods have been established. Among them, bacterial pure culture, physiological and biochemical detection, serological typing and phage typing have been widely used, but there are still time-consuming and low sensitivity. question

Method used

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  • Fluorescent quantitative PCR method for detecting toxin-producing streptococcus suis and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxin-producing streptococcus suis and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxin-producing streptococcus suis and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 detection primer amplification standard curve

[0051] 1. Microbial culture

[0052] Use alkaline peptone water as the culture medium for Streptococcus suis, and grow well in alkaline peptone water or plates with a pH of 8.8-9.0. The diameter of the colony on the alkaline plate is 2mm, round, smooth and transparent. The vigorously growing microbial samples were selected for subsequent experiments.

[0053] 2. Genomic DNA Extraction

[0054] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0055] 3. Production of standard plasmids for amplified products

[0056] DNA extracted from Streptococcus suis was used to amplify with the primers of rpoB gene, gene1, gene2, gene3 and gene4 respectively, after the amplified product was treated with A, it was ligated into the T vector using T4 ligase, and transduced After competent cells, the plasmid is amplified on a large scale. The primer sequences of the five genes are ...

Embodiment 2

[0071] Embodiment 2 Positive and negative sample detection situation

[0072] 1. Microbial culture

[0073] With embodiment 1.

[0074] 2. Genomic DNA Extraction

[0075] With embodiment 1.

[0076] 3. qPCR detection of 5 target genes

[0077] According to the instructions of BioRad iQ SYBR Green SuperMix, the qPCR amplification of the sample was carried out, and the Ct value reading corresponding to each pair of primers was obtained. Each qPCR reaction was set up with 3 biological repeats and 3 technical repeats, and the qPCR reaction system and amplification reaction conditions are shown in Table 5.

[0078] table 5

[0079]

[0080]

[0081] 4. Result Analysis

[0082] As shown in Table 6 and Table 7, the qPCR amplification results of 5 pairs of primers showed that Streptococcus suis showed positive results of rpoB gene, while other Streptococcus showed negative results. Therefore, for Streptococcus suis and other Streptococcus species, the detection of rpoB ge...

Embodiment 3

[0088] The sensitivity of embodiment 3 detection system

[0089] 1. Microbial culture

[0090] Use alkaline peptone water as the culture medium for Streptococcus suis, and grow well in alkaline peptone water or plates with a pH of 8.8-9.0. The diameter of the colony on the alkaline plate is 2mm, round, smooth and transparent. Select vigorously growing microbial samples for subsequent experiments, collect the bacteria and calculate the concentration, and dilute by 10 to get 10 bacteria / ml, 100 bacteria / ml, 1000 bacteria / ml, and 10000 bacteria / ml. Bacterial solutions with different concentration gradients were used for subsequent DNA extraction experiments.

[0091] 2. Genomic DNA Extraction

[0092] With embodiment 1.

[0093] 3. qPCR detection of 5 target genes

[0094] With embodiment 2.

[0095] 4. Result Analysis

[0096] As shown in Table 8 and Table 9, the qPCR amplification results of 5 pairs of primers showed that within the range from 10 bacteria to 10,000 bacte...

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Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) method for detecting toxin-producing streptococcus suis and a corresponding kit. According to the invention, specificgene detection is skillfully applied to distinguish streptococcus suis from other streptococcus and to distinguish toxic streptococcus and non-toxic streptococcus, and accurate bacterial genus information is obtained through comprehensive judgment. Compared with an existing mainstream detection kit, the kit for detecting the toxin-producing streptococcus suis has the advantages of being high in sensitivity, being rapid and convenient, being good in specificity, being rigorous and accurate in judgment and the like, and has good application prospects and market value.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a method for fluorescent quantitative PCR detection of toxigenic Streptococcus suis through specific genes and a corresponding detection kit. Background technique [0002] Streptococcus suis is a Gram-positive coccus with capsules. When grown on blood plate medium, a hemolytic ring is formed around the colony. S. suis can be classified as group D streptococci according to their cell wall antigenic composition. [0003] Streptococcus suis is a zoonotic pathogen. According to its capsule antigen (CPS), Streptococcus suis is divided into 35 serotypes (types 1-34, 1 / 2), of which 1, 2, 7 , Type 9 is the most pathogenic Streptococcus suis. The pathogenic factors of Streptococcus suis include capsular polysaccharide, lysozyme-releasing protein, extracellular factor, and hemolysin. The distribution of S. suis serotypes and virulence factor phenotypes had regio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12R1/46
CPCC12Q1/689C12Q1/6851C12Q2563/107C12Q2531/113
Inventor 曹亮吴胜标谢燕荣宁大亮石舟蒋华束文圣
Owner GUANGZHOU MICROCHIP BIOTECH CO LTD
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