Primer group for detecting mycoplasma pneumoniae, kit and method

A Mycoplasma pneumoniae and kit technology, applied in the fields of in vitro diagnosis and detection, and molecular biology, can solve the problems of high requirements for instrument platforms and operators, complex nucleic acid extraction and detection operations, limited use scenarios, etc., and achieve simple and enhanced identification. Convenience, the effect of improving the detection speed

Active Publication Date: 2021-05-25
SHANGHAI UPPER BIO TECH PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have obvious technical defects and application constraints. For example, real-time fluorescent quantitative PCR technology faces a series of problems such as complex nucleic acid extraction and detection operations, high requirements for instrument platforms and operators, high cost and limited use scenarios.

Method used

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  • Primer group for detecting mycoplasma pneumoniae, kit and method
  • Primer group for detecting mycoplasma pneumoniae, kit and method
  • Primer group for detecting mycoplasma pneumoniae, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1: LAMP method amplifies MP sequence

[0081] (1) Amplification reaction system:

[0082] 1.0mmol / L dNTP;

[0083] 0.2μmol / L MP outer primer pair;

[0084] 1.6μmol / L MP inner primer pair;

[0085] 0.4μmol / L MP loop primer pair;

[0086] 0.5U / μL Bst DNA polymerase;

[0087] 20mmol / L Tris-HCl (pH 8.8);

[0088] 10mmol / L KCl;

[0089] 10mmol / L (NH 4 ) 2 SO 4 ;

[0090] 0.1% Triton X-100;

[0091] 6mmol / L MgSO 4 ;

[0092] 0.5mol / L betaine;

[0093] 100X diluted SYBR Green.

[0094] (2) Amplification conditions: placed in a constant temperature nucleic acid amplification analyzer, amplified at 65°C for 20 minutes.

Embodiment 2

[0095] Embodiment 2: Detection of LAMP reaction sensitivity

[0096] At present, the mainstream molecular diagnostic products on the market are mostly based on QPCR. Although it is generally believed that the sensitivity performance is good (100-500copies / reaction), it is limited by the need for expensive variable temperature fluorescent quantitative PCR instruments and the slow heating and cooling speed of PCR cycles. And other disadvantages, the promotion has been limited. In contrast, the characteristics of isothermal LAMP technology just make up for the disadvantages of QPCR technology, but the literature reports and the sensitivity performance of LAMP products on the market are uneven. System optimization ensures that LAMP has high detection sensitivity.

[0097] Such as figure 1 , Table 2. The S-shaped curve in the figure is the fluorescence curve of the LAMP amplification experiment. The S-shaped curve means that the fluorescent signal increases. The essential princip...

Embodiment 3

[0101] Embodiment 3: detection of time-consuming reaction

[0102] Such as figure 1 , Table 2, and shown in Table 3, the primer set selected by the present invention can complete the LAMP amplification reaction within 20 minutes, which is much better than the time-consuming of 30-60 minutes of LAMP / mainstream QPCR products reported in the literature and on the market.

[0103] Table 3: Comparison of onset times of different MP primer sets

[0104]

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PUM

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Abstract

The invention discloses a primer group for detecting mycoplasma pneumoniae (MP), a kit and a method, the primer group comprises an MP outer primer pair, an MP inner primer pair and an MP loop primer pair, the sequence of the MP outer primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, the sequence of the MP inner primer pair is shown as SEQ ID NO: 3 and SEQ ID NO: 4, and the sequence of the MP loop primer pair is shown as SEQ ID NO: 5 and SEQ ID NO: 6. The primer group, the kit and the method provided by the invention have the advantages of high sensitivity (capable of reaching 100 copies / reaction), accurate detection, high detection speed and simple detection process.

Description

technical field [0001] The invention belongs to the fields of molecular biology, in vitro diagnosis and detection, and specifically relates to a primer set, a kit and a method for detecting mycoplasma pneumoniae. Background technique [0002] Mycoplasma pneumoniae is the main pathogen of community-acquired pneumonia and the main epidemic pathogen in winter and spring all over the world. It is highly contagious, and infants and young children are especially susceptible. The infection rate in the Chinese population is relatively high, and the incidence of infection has increased in recent years trend. Mycoplasma pneumoniae infection can cause pneumonia, and even erode extrapulmonary organs, and severe cases can lead to death. There are many types of respiratory pathogens, including viruses, bacteria, Mycoplasma pneumoniae, Chlamydia pneumoniae, and some protozoa and fungi. The preferred treatment drugs and treatment options for different pathogens are not the same. As far as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/35
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 张珂李威
Owner SHANGHAI UPPER BIO TECH PHARMA
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