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

A technique for streptococcus pyogenes and fluorescence quantification, applied in the field of molecular detection, can solve the problems of time-consuming and low sensitivity, and achieve the effects of wide application range, high sensitivity and stable detection 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

[0004] At present, many detection methods have been established for the differential diagnosis of Streptococcus pyogenes, among which smear staining microscopy, blood plate culture, physiological and biochemical detection, and Lamb's serological test have been widely used, but there are still time-consuming and low sensitivity. And other issues

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 detection primer amplification standard curve

[0049] 1. Microbial culture

[0050] Use alkaline peptone water as the culture medium of Streptococcus pyogenes, 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.

[0051] 2. Genomic DNA Extraction

[0052] Genomic DNA was extracted according to the instructions of Qiagen's QIAamp DNA Mini Kit.

[0053] 3. Production of standard plasmids for amplified products

[0054] Use the DNA extracted from Streptococcus pyogenes to amplify with the primers of rpoB gene, gene1, gene2, gene3 and gene4 respectively, after the amplified product is treated with A, it is 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 sequences of the...

Embodiment 2

[0068] Embodiment 2 Positive and negative sample detection situation

[0069] 1. Microbial culture

[0070] With embodiment 1.

[0071] 2. Genomic DNA Extraction

[0072] With embodiment 1.

[0073] 3. qPCR detection of 5 target genes

[0074] 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.

[0075] table 5

[0076]

[0077]

[0078] 4. Result Analysis

[0079] As shown in Table 6 and Table 7, the qPCR amplification results of 5 pairs of primers showed that Streptococcus pyogenes showed positive results of rpoB gene, while other Streptococcus showed negative results. Therefore, for Streptococcus pyogenes and other Streptococcus species, the detection of...

Embodiment 3

[0084] The sensitivity of embodiment 3 detection system

[0085] 1. Microbial culture

[0086] Use alkaline peptone water as the culture medium of Streptococcus pyogenes, 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.

[0087] 2. Genomic DNA Extraction

[0088] With embodiment 1.

[0089] 3. qPCR detection of 5 target genes

[0090] With embodiment 2.

[0091] 4. Result Analysis

[0092] The qPCR amplification results of 5 pairs of primers showed that within the range from 10 bacteria to 10000 bacteria, Streptococcus pyogenes sho...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting toxigenic Streptococcus pyogenes and a corresponding kit. According to the invention, specific gene detection is ingeniouslyapplied to distinguish Streptococcus pyogenes from other Streptococcus, and to distinguish toxigenic Streptococcus from non-toxigenic Streptococcus, and accurate bacterial genus information is obtained through comprehensive judgment. Compared with existing mainstream detection kits, the kit for detecting the toxigenic Streptococcus pyogenes 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 pyogenes through specific genes and a corresponding detection kit. Background technique [0002] Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is β-hemolytic, sensitive to bacitracin, positive for PYR, has Langer’s Group A antigen, and forms large colonies on blood agar . Streptococcus pyogenes is spherical or oval under the microscope, with a diameter of about 0.5-1.0 μm, arranged in chains with different chain lengths, and it is easy to form long chains in liquid medium. The bacterium has no spores and no flagella. Most strains formed hyaluronic acid capsules in the early stage of culture (2-4 hours). Streptococcus pyogenes is easily stained by common basic dyes. Newly isolated strains from the lesion are Gram-positive, and old bacteria or bacteria that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/14C12N15/11C12R1/46
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 曹亮吴胜标谢燕荣吴海滨宁大亮石舟蒋华束文圣
Owner GUANGZHOU MICROCHIP BIOTECH CO LTD
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