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Fluorescent quantitative PCR method for detecting toxigenic Streptococcus agalactiae and corresponding kit

A Streptococcus lactis, fluorescence quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of low sensitivity, time-consuming, etc. The effect of stable test results

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

These methods are not only time-consuming and low in sensitivity, but also the reaction is not specific to the group B strains of Streptococcus agalactiae, and some streptococci in groups C, F, and G may also have positive results.

Method used

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  • Fluorescent quantitative PCR method for detecting toxigenic Streptococcus agalactiae and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxigenic Streptococcus agalactiae and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxigenic Streptococcus agalactiae and corresponding kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 detection primer amplification standard curve

[0050] 1. Microbial culture

[0051] Use alkaline peptone water as the culture medium for Streptococcus agalactiae, and grow well in alkaline peptone water or plates with a pH of 8.8 to 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.

[0052] 2. Genomic DNA Extraction

[0053] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0054] 3. Production of standard plasmids for amplified products

[0055] DNA extracted from Streptococcus agalactiae was used to amplify with the primers of rpoB gene, gene1, gene2, gene3 and gene4 respectively, and after the amplified product was treated with A, it was ligated into the T vector using T4 ligase, and passed After induction of competent cells, the plasmids were amplified on a large scale. The primer s...

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 agalactiae showed positive results of rpoB gene, while other Streptococcus showed negative results. Therefore, for Streptococcus agalactiae and other Streptococci, the detection of rpoB...

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 agalactiae, and grow well in alkaline peptone water or plates with a pH of 8.8 to 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,...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting toxigenic Streptococcus agalactiae and a corresponding kit. According to the invention, specific gene detection is ingeniously applied to distinguish Streptococcus agalactiae from other Streptococcus and to distinguish toxigenic Streptococcus agalactiae from non-toxigenic Streptococcus agalactiae, and accurate bacteria genus information is obtained through comprehensive judgment. Compared with existing mainstream detection kits, the kit for detecting the toxigenic Streptococcus agalactiae has the advantages of being high in sensitivity, rapid, convenient, good in specificity, 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 agalactiae through specific genes and a corresponding detection kit. Background technique [0002] Streptococcus agalactiae is a Gram-positive coccus arranged in pairs or short chains. It is a β-hemolytic, catalase-negative, VP-test-positive facultative anaerobic bacterium. Streptococcus agalactiae, also known as group B streptococcus, can be divided into 10 serotypes (Ia, Ib, II–IX) according to the immunological activity of its polysaccharide capsule. [0003] In general, Streptococcus agalactiae is a harmless commensal bacterium that is part of the human microbiota that colonizes the gastrointestinal and urogenital tracts of 30% of healthy human adults when When the immune function of the human body is reduced, it will provide opportunities for Streptococcus agalactiae infe...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
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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