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Fluorescent quantitative PCR method for detecting toxigenic chlamydia pneumoniae and corresponding kit

A Chlamydia pneumoniae, fluorescent quantitative technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the difficult to distinguish bacterial, viral Mycoplasma pneumoniae or Chlamydia pneumoniae infection, test To solve problems such as complicated process and difficult clinical medication, to achieve stable test results, wide application range, rigorous and accurate results

Pending Publication Date: 2020-09-18
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

[0004] Clinically based on the symptoms and signs of patients with Chlamydia pneumoniae infection, it is difficult to distinguish bacterial, viral, Mycoplasma pneumoniae or Chlamydia pneumoniae infection, so that there are difficulties in clinical medication, and generally can only rely on laboratories for etiological detection through human body fluid samples
Commonly used microbiological inspection methods include isolation culture and serological detection, which have the characteristics of poor sensitivity and complicated test process

Method used

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  • Fluorescent quantitative PCR method for detecting toxigenic chlamydia pneumoniae and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxigenic chlamydia pneumoniae and corresponding kit
  • Fluorescent quantitative PCR method for detecting toxigenic chlamydia pneumoniae and corresponding kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 detection primer amplification standard curve

[0049] 1. Microbial culture

[0050] Alkaline peptone water is used as the culture medium for toxigenic Vibrio vulnificus, and it grows 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 Chlamydia pneumoniae to amplify with the primers of rpoB gene, gene1 gene, gene2 gene and gene4 gene respectively. After competent cells, the plasmid is amplified on a large scale. The primer sequences of the five genes are shown in SEQ ID NO: 1-10, and the details are shown in Table 2.

[0055] Table 2

[0...

Embodiment 2

[0067] Embodiment 2 Positive and negative sample detection situation

[0068] 1. Microbial culture

[0069] With embodiment 1.

[0070] 2. Genomic DNA Extraction

[0071] With embodiment 1.

[0072] 3. qPCR detection of 5 target genes

[0073] 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.

[0074] table 5

[0075]

[0076]

[0077] 4. Result Analysis

[0078] The qPCR amplification results of 5 pairs of primers showed that Chlamydia pneumoniae showed positive results of one or more genes of rpoB gene, gene1 gene, gene2 gene and gene3 gene, while other Chlamydia species showed negative results. Therefore, for Chlamydia pneumoniae and other Chlamydia ge...

Embodiment 3

[0082] The sensitivity of embodiment 3 detection system

[0083] 1. Microbial culture

[0084] Use alkaline peptone water as the culture medium for Vibrio vulnificus, 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.

[0085] 2. Genomic DNA Extraction

[0086] With embodiment 1.

[0087] 3. qPCR detection of 5 target genes

[0088] With embodiment 2.

[0089] 4. Result Analysis

[0090] The qPCR amplification results of 5 pairs of primers showed that within the range from 10 bacteria to 10,000 bacteria, Chlamydia pneumoniae showed p...

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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting toxigenic chlamydia pneumoniae and a kit. The method ingeniously applies specific gene detection to distinguish chlamydia pneumoniae from other chlamydia and toxigenic and non-toxigenic chlamydia pneumoniae, and obtains accurate bacterial information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting toxigenic chlamydia pneumoniae has the advantages of high sensitivity, quickness, convenience, good specificity, rigorous and accurate judgment and the like, and hasgood application prospect and market value.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a detection method of fluorescent quantitative PCR for toxigenic chlamydia pneumoniae through specific genes and a corresponding detection kit. Background technique [0002] Chlamydia pneumoniae has no motility and can perform limited metabolic activities independently. The chromosome of Chlamydia pneumoniae has two kinds of nucleic acids, DNA and RNA, which can only replicate and reproduce in the cell. The developmental cycle is divided into two stages: the protosome and the reticulum. The average diameter of the protoplasma was 0.38nm, and it was pear-shaped under the electron microscope, with clear periplasmic gaps. There was no plasmid DNA in the protoplasma. Giemsa stained purple, Macchiavello stained red. Protomers are highly infectious, relatively stable outside the host cell, and have no ability to reproduce. After entering the susceptible cells...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2545/114C12Q2563/107
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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