Fluorescent quantitative PCR method for detecting toxin-producing neisseria meningitidis and corresponding kit

A technology of Neisseria and Neisseria, which is applied in the field of molecular detection, can solve the problems of low detection rate of separation and culture method, low sensitivity of separation and culture method, and reduced reliability of diagnosis results, and achieves detection results. Stable, wide range of applications, to achieve the effect of correct detection

Pending Publication Date: 2020-08-14
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection rate of the isolation and culture method is low, and it takes at least 2 to 3 days to confirm the infection, which is extremely unfavorable to the timely treatment of the disease.
In

Method used

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  • Fluorescent quantitative PCR method for detecting toxin-producing neisseria meningitidis and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxin-producing neisseria meningitidis and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxin-producing neisseria meningitidis and corresponding kit

Examples

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 Neisseria meningitidis, 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 Neisseria meningitidis was used to amplify with primers of rpoB gene, gene1, gene2, gene3, and gene4 respectively, and the amplified products were treated with A, and then ligated into the T vector using T4 ligase. And after transducing competent cells, the plasmid was amplified on a large scale. The primer sequences of the fiv...

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] 4. Result Analysis

[0078] As shown in Table 6 and Table 7, the qPCR amplification results of 5 pairs of primers show that Neisseria meningitidis presents positive results of one or more genes of rpoB gene, gene1, gene2 and gene3, while other cocci have all positive results. Shows a negative result. Therefore, for Neisseria meningiti...

Embodiment 3

[0084] The sensitivity of embodiment 3 detection system

[0085] 1. Microbial culture

[0086] Use alkaline peptone water as the culture medium for Neisseria meningitidis, 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.

[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] As shown in Table 8 and Table 9 below, the qPCR amplification results of 5 pairs of primers show that within the range from 10 bacteria to 1...

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Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) method for detecting toxin-producing neisseria meningitidis and a corresponding kit. According to the invention, specific gene detection is ingeniously applied to distinguish neisseria meningitidis from other cocci and to distinguish toxic and non-toxic cocci, and accurate bacteria genus information is obtained through comprehensive judgment. Compared with an existing mainstream detection kit, the kit for detecting the toxin-producing neisseria meningitidis has the advantages of being high in sensitivity, beingrapid and convenient to use, 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 performing fluorescence quantitative PCR detection on toxin-producing Neisseria meningitidis through specific genes and a corresponding detection kit. Background technique [0002] Neisseria meningitidis (Neisseria meningitidis) is a Gram-negative aerobic diplococcus, often in double arrangement, with a diameter of about 0.8 μm. Neisseria meningitidis breaks down sugars to produce acid but not gas. Oxidase test was positive. Neisseria meningitidis can produce autolytic enzymes, if not transplanted in time during artificial culture, the bacteria will autolyze after a few days. The main antigens of Neisseria meningitidis include capsular polysaccharide antigen, outer membrane protein and lipopolysaccharide antigen. According to the antigenicity of capsular polysaccharides, Neisseria meningitidis can be divided into at least 13 serogroups. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/36
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 曹亮张智闵连政汉张新帅王萌萌王璋赵亮郝祎琪蒋华束文圣
Owner 广东美格基因科技有限公司
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