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Target sequence used for detecting mycoplasma pnoumoniae and reagent box

A Mycoplasma pneumoniae and kit technology, applied in the field of molecular biology and nucleic acid detection, can solve the problems of little clinical help and slow growth of Mycoplasma pneumoniae

Active Publication Date: 2006-01-25
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because Mycoplasma pneumoniae grows slowly, it takes 2 to 3 weeks to identify Mycoplasma pneumoniae by culturing patient specimens, so the culture method is not very helpful clinically

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Isolation of Mycoplasma pneumoniae DNA for PCR Analysis

[0057] In order to isolate M. pneumoniae DNA from test samples for PCR detection, simple methods that do not interfere with PCR must be used. First, rinse the clinical sample (throat swab) with sterilized physiological saline, centrifuge the rinsing solution at 15,000 rpm for 15 minutes and discard the supernatant, so that the mycoplasma pneumoniae that may be contained in the rinsing solution is collected. After adding DNA lysate to the precipitate, shake and mix well, and bathe in boiling water at 100°C for 15 minutes to lyse Mycoplasma pneumoniae and release DNA. Finally, centrifuge at 15,000 rpm for 15 minutes, dissolve the DNA into the supernatant, and take the supernatant as the Mycoplasma pneumoniae DNA for PCR analysis.

Embodiment 2

[0058] The preparation of embodiment 2 detection kits

[0059] A fluorescent PCR kit for rapid quantitative detection of Mycoplasma pneumoniae is prepared by a conventional method, and the kit includes a) DNA lysate, b) fluorescent quantitative reaction solution, c) quantitative calibrator, d) positive control substance, e) negative control Taste. in:

[0060] 1) Fluorescence quantitative reaction solution contains PCR buffer, dNTPs solution, Taq DNA polymerase, sterile double distilled water, MgCl 2 Solution, the primer sequences for specific amplification of P1 cell adhesion protein gene genetic markers are SEQ ID NO: 3 and SEQ ID NO: 4, the fluorescent probe sequence is SEQ ID NO: 5, and the 5' end of the fluorescent probe is labeled The fluorescent reporter group is FAM, and the fluorescent quencher group labeled at the 3' end is TAMRA. (Amplification product size is 160bp)

[0061] Specifically, the fluorescence quantitative reaction solution contains 50mM KCl, 10mM T...

Embodiment 3

[0065] Embodiment 3 carries out polymerase chain reaction with mycoplasma pneumoniae

[0066] Take respectively 26ul of the fluorescent reaction solution in Example 2 and add them to different PCR reaction tubes, add 4ul of the DNA and quantitative calibrator obtained in Example 1 to the fluorescent quantitative reaction solution, and add a total volume of 30ul in the real-time fluorescent quantitative PCR instrument (Switzerland Roche Company) began to amplify and detect. The amplification parameters were set as denaturation temperature at 93°C for 10 seconds, annealing temperature at 55°C for 10 seconds, and extension temperature at 72°C for 20 seconds, and 40 cycles of PCR amplification were performed. Fluorescence detection was programmed to detect at a wavelength of 530 nm at the end of the annealing step of each cycle.

[0067] Sample to be tested

[0068]This shows that in the kit of the present invention, it can be 6 Mycoplasma pneumoniae can be amplified...

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PUM

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Abstract

The invention supplies a method to rapidly test the genetic marker of Mycoplasma pneumoniae. It supplies the nucleotide sequence of Pl CytadhesinGene and primer and probe that is designed based on the sequence. The invention also supplies the method to rapidly testing the Mycoplasma pneumoniae by using the primer.

Description

technical field [0001] The present invention relates to the fields of molecular biology and nucleic acid detection, in particular to a method for amplifying P1 cell adhesion protein gene (P1 Cytadhesin Gene) by polymerase chain reaction to specifically detect Mycoplasma pneumoniae in clinical samples with high sensitivity and its utilization The method obtains a real-time fluorescent PCR detection kit. Background technique [0002] Mycoplasma pneumonia is a very serious infectious disease. In pneumonia, mycoplasma pneumonia accounts for about 15% to 20%, and causes many complications. Mycoplasma pneumonia is an acute lung infection caused by Mycoplasma pneumoniae. Mycoplasma pneumoniae can invade the respiratory system of people of any age, and it can also cause diseases to many other systems of the human body, with complex clinical manifestations. In recent years, the number of patients infected with Mycoplasma pneumoniae has increased, so the harm of Mycoplasma pneumonia...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李超周煜夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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