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Fluorescent quantitative PCR (Polymerase Chain Reaction) primers for detecting mycoplasma pneumoniae (MP) and application thereof

A technology of mycoplasma pneumoniae and primer pairs, which is applied in the field of pathogen detection, can solve the problems of clinical application limitations, high false positive rate, continuous positive and other problems, and achieve good detection effect and application value, high sensitivity and strong specificity

Active Publication Date: 2014-04-23
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Antibody detection methods for indirect diagnosis of MP infection have been widely used in clinical detection, but the sensitivity of these methods is not ideal, and there is a persistent positive phenomenon after MP infection
PCR diagnostic technology has already been applied to the diagnosis of MP infection in foreign countries, but due to the high false positive rate due to the easy contamination during the ordinary PCR operation, the clinical application is limited

Method used

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  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primers for detecting mycoplasma pneumoniae (MP) and application thereof
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primers for detecting mycoplasma pneumoniae (MP) and application thereof
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) primers for detecting mycoplasma pneumoniae (MP) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the acquisition of the fluorescent PCR primer that is used to detect Mycoplasma pneumoniae

[0031] 1. Design and synthesis of primer pairs

[0032] According to the genome DNA sequence (GenBank sequence number: CP002077) of the human Mycoplasma pneumoniae international standard strain FH (ATCC15531) published on GenBank, the 16S rRNA (GenBank sequence number: AF132740) and ATPase gene sequence (GenBank sequence number: AF132740) of the strain (GenBank The sequence number: U43738) was used as a template, and the primer design software Primer Primer5.0 was used to design primers. Avoid areas with high homology with other pathogens, manually adjust the parameters, the size of the amplification product is in the range of 150-300bp, and the Tm value of the primers is in the range of 54-64°C. A total of 20 pairs of primers were designed and selected, among which the 16S rRNA sequence 6 pairs of primers, 14 pairs of primers for ATPase sequence. The obtained prim...

Embodiment 2

[0045] Embodiment 2, the specificity detection of the fluorescent PCR primer that is used to detect Mycoplasma pneumoniae and the determination of the minimum detection limit

[0046] In this example, the international standard strain of Mycoplasma pneumoniae FH (ATCC15531), four kinds of mycoplasma pathogens and 11 kinds of common respiratory tract bacteria and fungi were used to detect the specificity of the three primer pairs obtained in Example 1. Among them, four kinds of Mycoplasma pathogens are: Mycoplasma genitalium (ATCC33530), Mycoplasma hominis (ATCC23114), Ureaplasma urealyticum (ATCC27618) and Mycoplasma pirilum (ATCC25960); 11 common bacteria and fungi in the respiratory tract are: Haemophilus influenzae (hin) (ATCC49247), Streptococcus A (gas) (ATCC19615), Staphylococcus epidermidis (sep) (ATCC12228), Staphylococcus aureus (sau) (ATCC25923), Escherichia coli (eco) (ATCC25922) , Klebsiella pneumoniae (kpn) (ATCC700603), Moraxella catarrhalis (bca) (ATCC25238), Ac...

Embodiment 3

[0057] Embodiment 3, the clinical sample verification of the fluorescent PCR primer that is used to detect Mycoplasma pneumoniae

[0058] In this example, the inventor retrospectively analyzed the MP-related test results of 100 patients with community-acquired pneumonia (age ≥ 14 years old) in the fever clinic, and the MP-IgG antibody in the double serum was increased by 4 times or more as MP infection The "gold standard" of diagnosis, respectively evaluate the MP FQ-PCR product of Sun Yat-Sen University Daan Gene Co., Ltd. (catalogue number: DA-B064), the real-time fluorescent quantitative PCR method for MPP1 using the primers obtained in Example 1, and the detection of IgM antibody The detection rate, sensitivity, specificity, positive predictive value, negative predictive value and positive likelihood ratio of the method and culture method.

[0059] At the first outpatient visit (acute stage of infection) of each patient, throat swabs were collected and stored in 2ml of nor...

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Abstract

The invention discloses fluorescent quantitative PCR (Polymerase Chain Reaction) primers for detecting mycoplasma pneumoniae (MP) and application thereof. A primer pair provided by the invention is specific to a 16SrRNA gene or ATPase gene of MP and can be a primer pair 1 (shown by a sequence 1 and a sequence 2) or a primer pair 2 (shown by a sequence 3 and a sequence 4) or a primer pair 3 (shown by a sequence 5 and a sequence 6). Shown by experiments, compared with other commercialized detection primers, IgM (Immunoglobulin M) antibody detection methods and culture methods, a method in which the primer pair provided by the invention can be used for carrying out fluorescent quantitative PCR detection on test samples has the advantage that the MP can be specifically detected without being interfered by four kinds of common pathogenic mycoplasmas which have a relatively close genetic relationship with the MP, as well as common respiratory tract bacteria and fungal pathogenic bacteria. The method can be used for qualitatively detecting the MP and detecting the MP quantitatively very well, is a quantitative PCR detection technology which is quick, is strong in specificity and high in sensitivity and is suitable for clinically detecting the MP of all strains, and has good detection effect and application value in clinical detection.

Description

technical field [0001] The invention belongs to the field of pathogen detection, and relates to a fluorescent quantitative PCR primer for detecting mycoplasma pneumoniae and an application thereof. Background technique [0002] Mycoplasma pneumoniae (MP) is a very important pathogen in respiratory tract infections, including community-acquired pneumonia (CAP). Mycoplasma pneumoniae pneumonia widely exists in the world, and most of them are sporadic cases. There is a regional epidemic in about 3-6 years, and the epidemic time can be as long as 1 year. The incidence rate in the epidemic year can reach several times that in the non-epidemic year. The incidence is concentrated in densely populated environments such as schools, kindergartens, and the military. A recent global survey on the etiology of CAP including Asia showed that Mycoplasma pneumoniae pneumonia accounted for 12% of CAP and more than 50% of all CAP caused by atypical pathogen infection. Compared with most fore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2561/113C12Q2563/107
Inventor 曹彬曲久鑫高宇辉
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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