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Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia

A Mycoplasma pneumoniae and probe technology, applied in the field of molecular biology, can solve the problems of lack of sensitivity and specificity, comparison, etc., and achieve the effects of increasing the positive rate, simplifying the procedure, and shortening the detection cycle

Active Publication Date: 2011-11-02
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no p1 gene sequence of domestic strains in the NCBI database, although these methods are also used in China, there is no real comparison of the sensitivity and specificity of the detection of Mycoplasma pneumoniae strains in China

Method used

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  • Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia
  • Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia
  • Target sequence, primer, probe and kit for detecting Mycoplasma pneumonia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The p1 gene of 60 clinical isolates of Mycoplasma pneumoniae was amplified, sequenced, sequence compared and probe and primer design.

[0045] Common PCR primer sequences are:

[0046] P1primer-F: 5'-ATGCACCAAACCAAAAAAACTGCCT-3'

[0047] P1primer-R: 5'-CTAAGCGGGTTTTTTAGGTGGTTGC-3'

[0048] Use TAKARALA Taq kit (DRR200A) to amplify, reaction system:

[0049]

[0050] Amplification conditions:

[0051]

[0052] The amplified products were sequenced and spliced ​​by Huada Genomics (BGI). The spliced ​​and manually proofreaded 60 p1 gene sequences and all the p1 gene sequences reported in the NCBI database were compared using Vector NTI Suite 6 software to find out the Conserved area. The p1 gene is a unique functional gene sequence of Mycoplasma pneumoniae itself. By comparing the p1 gene sequences of 60 domestic strains of Mycoplasma pneumoniae with the reported p1 gene sequences in the NCBI database, a completely conserved region of nucleotides 262bp was found:...

Embodiment 2

[0056] Optimization of Experimental Parameters for Real-time Fluorescent Quantitative PCR

[0057] (1) Optimization of magnesium ion concentration in the system: MgCl in the system 2 Increase from 0.5ul to 4ul in sequence, each increment is 0.5ul, and three parallel samples are made for each concentration gradient. ResultMgCl 2 The amount added is 2ul (MgCl 2 When the final concentration is 4mM), the amplification effect of the system is the best ( Figure 1a ).

[0058] (2) System annealing temperature optimization: the system annealing temperature was changed from 55°C to 65°C, and the results showed that the amplification effect of the system around 59°C was the best ( Figure 1b )

[0059] (3) Optimization of MpP1 real-time fluorescent quantitative PCR amplification system and amplification conditions

[0060]

[0061] Amplification conditions:

[0062]

Embodiment 3

[0064] Preparation of system standard curve

[0065] The real-time PCR standard curve of the MpP1 system was drawn with the value of 1 g of the standard concentration template as the abscissa and the corresponding Ct value as the ordinate. The results show that: when the amount of positive template is in the range of 8.1fg-8.1ng, its logarithmic value has a very good correlation with the Ct value (R 2 = 0.996). See figure 2 .

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Abstract

The invention provides a genetic marker, real-time fluorescence quantitative PCR (polymerase chain reaction) primers and probe for detecting Mycoplasma pneumonia, by sequencing and comparing of Mycoplasma pneumonia genes, which have the nucleotide sequences shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. The invention also provides a method and kit for quantitatively detecting Mycoplasma pneumonia. The detection method has the advantages of accuracy in detection, high sensitivity and strong specificity, is simple and rapid in operation, and is superior in sample detection capacity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a target sequence for detecting mycoplasma pneumoniae, a fluorescent quantitative PCR primer and a probe, and also relates to a method and a kit for detecting mycoplasma pneumoniae by using the target sequence. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae) is one of the important pathogens causing respiratory tract infection, 10% to 40% of community-acquired pneumonia is caused by Mycoplasma pneumoniae infection. At present, domestic clinical detection techniques for Mycoplasma pneumoniae are limited, resulting in the abuse of antibiotics and increasing the medical burden. Due to the complicated and time-consuming technology of isolation and culture of Mycoplasma pneumoniae, its detection technology mainly relies on serological detection and nucleic acid detection. Fluorescent PCR detection technology that has emerged in recent years has been more a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 赵飞张建中
Owner ICDC CHINA CDC
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