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Primers and kit for detecting mycoplasma pneumoniae

A technology of mycoplasma pneumoniae and kits, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of shortened reaction time, achieve shortened detection time, high sensitivity, and improve detection efficiency Effect

Inactive Publication Date: 2021-04-16
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the development of PCR technology in the future, the reaction time will also be shortened, and the advantages of LAMP will not be as obvious as they are now

Method used

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  • Primers and kit for detecting mycoplasma pneumoniae
  • Primers and kit for detecting mycoplasma pneumoniae
  • Primers and kit for detecting mycoplasma pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and screening of fluorescent ring-mediated isothermal amplification primer set for detection of Mycoplasma pneumoniae in the present invention

[0042] The primers in the present invention are designed and prepared according to the following method: using the Mycoplasma pneumoniae SDC1 gene sequence (GenBank accession No. M35024) as a template to design outer primers (F3 and B3), inner primers (FIP and BIP), and two accelerated amplification The loop primers for the amplification reaction were artificially synthesized.

[0043] The optimization and screening process of the primer set in the present invention:

[0044]In order to develop amplification primers suitable for the fluorescent LAMP system that meet clinical needs, dozens of primers with different sequences were designed for different amplification fragments, and four primers F3, B3, FIP and BIP of these primary screening primers were first synthesized. Then use standard strain DNA for amplifi...

Embodiment 2

[0073] Primer set sensitivity and specificity experiment in embodiment 2 of the present invention

[0074] After the screening in Example 1 above, the set of primers MP2 was initially selected for subsequent verification experiments.

[0075] One. Sensitivity detection of primer sets in the present invention:

[0076] 1. Detection method: extract the genomic DNA of MP standard strain FH strain (ATCC15531), and perform 10-fold equivalent dilution with TE buffer, as a template in the fluorescent LAMP reaction system. Each reaction system contains 0.6, 6, 60, 600, 6000, 60000 copies of DNA template of MP respectively. The experiment was repeated three times.

[0077] 2. Sensitivity test results of the primer set in the present invention:

[0078] The DNA extracted and purified from the MP standard strain FH culture solution was diluted 10 times and used in the fluorescent LAMP reaction system, so that each reaction system contained 0.6, 6, 60, 600, 6000, 60000 copies respectiv...

Embodiment 3

[0083] Application of the primer set in the embodiment 3 of the present invention in the detection of Mycoplasma pneumoniae

[0084] Utilize the kit in the present invention to carry out detection verification to 247 clinical specimens.

[0085] 1. Source of samples: 247 outpatient and inpatient pediatric patients in a tertiary hospital were selected. Pharyngeal swab specimens were collected from the patients. The swabs were placed in 2ml MP transfer medium and frozen at -20°C for later use.

[0086] 2. Inclusion criteria: Fever (body temperature ≥ 38°C), cough or sore throat and other symptoms of respiratory tract infection, the course of the disease is within 7 days, mainly community-acquired pneumonia or mycoplasma pneumonia.

[0087] 3. Exclusion criteria: patients with pulmonary tuberculosis and lung tumors, non-infectious pulmonary interstitial pneumonia, pulmonary edema, atelectasis, pulmonary embolism, pulmonary eosinophilic infiltration, and immunocompromised patients...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer group for detecting mycoplasma pneumoniae. The loop-mediated isothermal amplification primer group is composed of the following three pairs of primers: MP2-F3 and MP2-B3, MP2-FIP and MP2-BIP, and MP2-LF and MP2-LP, wherein the nucleotide sequence of the MP2-F3 is shown as SEQ ID No. 1, the nucleotide sequence of the MP2-B3 is shown as SEQ ID No. 2, the nucleotide sequence of the MP2-FIP is shown as SEQ ID No. 3, the nucleotide sequence of the MP2-BIP is shown as SEQ ID No. 4, the nucleotide sequence of the MP2-LF is shown as SEQ ID No. 5, and the nucleotide sequence of the MP2-LP is shown as SEQ ID No. 6. The primer group provided by the invention can significantly improve detection efficiency, shortens detection time to be within 30 minutes, a decrease by a half compared with 60 minutes or more needed in existing loop-mediated isothermal amplification methods, can be used for rapid detection of mycoplasma pneumoniae infection, provides powerful help for clinical early diagnosis and treatment, and buys time for timely isolation of infectors and control of infection diffusion.

Description

technical field [0001] The invention relates to a primer set for detecting mycoplasma pneumoniae, in particular to a fluorescent ring-mediated isothermal amplification primer set and a kit for detecting mycoplasma pneumoniae. Background technique [0002] Mycoplasma pneumoniae (MP for short) is the main pathogen that causes Mycoplasma pneumoniae pneumonia (MPP), and Mycoplasma pneumoniae pneumonia accounts for about 20-30% of all pneumonias in the world, and there is an increasing trend year by year. MP is also the main pathogen causing respiratory tract infection in children. MP infection not only causes upper and lower respiratory tract diseases, but also affects other systems of the human body, causing liver function damage, brain damage, myocarditis, arthritis and other extrapulmonary complications, ranging from mild to severe, and even life-threatening serious complications . [0003] Laboratory diagnosis is crucial for the diagnosis and treatment of patients with sus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
Inventor 李丹辛德莉
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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