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Method for detecting resistant mutant of mycoplasma pneumoniae

A technology of mycoplasma pneumoniae and drug-resistant mutations, applied in the fields of biology and medicine, can solve the problems of children with abnormal cartilage development, and achieve the effect of high specificity and easy operation

Inactive Publication Date: 2014-07-09
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Tetracyclines are restricted in late pregnancy, neonates, and children under 8 years of age because they can cause tooth staining. Fluoroquinolones may cause cartilage dysplasia and should not be used in children
If the RNA and adjacent bases of the probe do not match the template, RNase H will not be able to cut the probe, so cycling probe technology is a highly specific detection method for single-base mutations. There is no report yet

Method used

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  • Method for detecting resistant mutant of mycoplasma pneumoniae
  • Method for detecting resistant mutant of mycoplasma pneumoniae
  • Method for detecting resistant mutant of mycoplasma pneumoniae

Examples

Experimental program
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Embodiment 1

[0030] Embodiment 1: the specificity analysis of PCR primer of the present invention

[0031] Use Tiangen Bacteria Genomic DNA Extraction Kit (product number: DP302-02) to extract Mycoplasma pneumoniae standard strain (ATCC15531), Mycoplasma pneumoniae standard strain (ATCC29342), Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC700603) , Staphylococcus aureus (ATCC25923), Streptococcus pneumoniae (ATCC49619), Mycoplasma pneumoniae macrolide-sensitive clinical strains, Mycoplasma pneumoniae 2063 mutation-resistant clinical strains, Mycoplasma pneumoniae 2064 mutation-resistant clinical strains of genomic DNA It is the amplification template (extraction time is about 25 minutes). The amplification system was prepared according to the instructions with specific upstream and downstream primers for Mycoplasma pneumoniae and Takara PCR amplification reagent (product number: DR100A). The amplification conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at ...

Embodiment 2

[0032] Embodiment 2: Mycoplasma pneumoniae clinical isolate of the present invention

[0033] The PCR amplification products of the Mycoplasma pneumoniae standard strain (ATCC15531, a macrolide-sensitive strain), the 2063 mutant strain and the 2064 mutant strain were gel-cut and purified, inserted into the PCR product cloning vector, confirmed by sequencing, purified, and read Calculate the plasmid copy number after the OD value, and dilute this vector to 10 5 Copy / ml Take 1 microliter for each PCR (about 10 2 copy) was used as a positive control, and the negative control was Escherichia coli genomic DNA at the same concentration. The above, downstream primers, non-mutation-sensitive strain detection probe, 2063 mutation-resistant strain probe, 2064 mutation-resistant strain probe and Takara's CycleavePCR TM The Core Kit (product number: DCY501) was used to prepare the PCR reaction system according to the instructions. The concentrations of the three probes were optimized t...

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Abstract

The invention belongs to the fields of biology and medicines and particularly relates to a method for detecting a resistant mutant of mycoplasma pneumoniae. By adopting Real-time PCR (Polymerase Chain Reaction) and a cycling probe technology for distinguishing the difference of monobases and searching a specific primer aiming at the mycoplasma pneumoniae in an upstream and a downstream sequence of 2063 bit / 2064 bit of the mycoplasma pneumoniae 23SrRNA gene, specific cycling probes aiming at mutation-free sensitive strains, 2063-bit mutant drug-resistant strains and 2064-bit mutant drug-resistant strains can be respectively designed according to the difference of the bases at the 2063 bit / 2064 bit of the mutation-free sensitive strains and mutant drug-resistant 23SrRNA gene of the mycoplasma pneumoniae; the PCR amplification can be carried out by using the specific primer of the mycoplasma pneumoniae; and the fluorescence intensity change can be read by a Real-time PCR thermal cycler in real time to determine the mutant types of samples to be detected. The invention can correctly distinguish the mutation-free sensitive strains from the mutant drug-resistant strains of clinical separation strains of the mycoplasma pneumoniae. The specificity is 100 percent and the sensitivity can reach 102copy / PCR reaction.

Description

technical field [0001] The invention belongs to the fields of biology and medicine, and relates to a method for testing drug-resistant pathogenic microorganisms, in particular to a method for detecting drug-resistant mutations of Mycoplasma pneumoniae. In particular, a method for detecting Mycoplasma pneumoniae and its macrolide resistance mutations. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) belongs to Mollusca class, Mycoplasma family, Mycoplasma genus. It was first isolated from the pleural fluid by Chanock et al. in 1962. Mycoplasma pneumoniae can cause bronchitis and atypical pneumonia. Foy reported that between 1962 and 1975, 15% to 20% of community-acquired pneumonia was caused by Mycoplasma pneumoniae (Clin Infect Dis. 697). A recent epidemiological survey of adult community-acquired pneumonia in Asia showed that Mycoplasma pneumoniae pneumonia accounted for about 12.2% (Int J Infect Dis. 2005, 9: 144). Therefore, Mycoplasma ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 徐晓刚刘杨王明贵
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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