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Fluorescent PCR (polymerase chain reaction) primer, probe and detecting kit for detecting mycoplasmas genitalium

A technology of mycoplasma genitalium and fluorescent probe, applied in the field of molecular biology

Active Publication Date: 2018-08-17
西安博睿康宁生物医学中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The target sites of the existing fluorescent PCR detection for the detection of Mycoplasma genitalium are all concentrated in 16S rDNA, mg219, MgPa and other gene sequences, and its detection sensitivity ranges from a few copies to dozens of copies

Method used

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  • Fluorescent PCR (polymerase chain reaction) primer, probe and detecting kit for detecting mycoplasmas genitalium
  • Fluorescent PCR (polymerase chain reaction) primer, probe and detecting kit for detecting mycoplasmas genitalium
  • Fluorescent PCR (polymerase chain reaction) primer, probe and detecting kit for detecting mycoplasmas genitalium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 is used to detect the screening and determination of the specific repeat sequence of Mycoplasma genitalium

[0042] All MgPar repeat sequences of Mycoplasma genitalium in the NCBI database were aligned using Vector NTI Suite 6 software to find out the conserved regions. A 117bp highly conserved region of nucleotides in the repeated sequence was found, as shown in SEQ ID NO.1. The sequence alignment results showed that at least 6 of the above-mentioned 9 repeated sequences of each strain of Mycoplasma genitalium contained in the selected region The nucleic acid sequence of the underlined part (including single base polymorphism, which can be achieved by designing merged bases but the merger of base polymorphisms) is the most stable core region in the conserved region ( figure 1 ). Therefore, the core conserved sequence can be used as a fluorescent PCR detection target sequence.

Embodiment 2

[0043] Embodiment 2 detects the primer and the probe of the specific repetitive sequence of Mycoplasma genitalium

[0044] For the target sequence SEQ ID NO.1 used to amplify Mycoplasma genitalium determined in Example 1, the inventor designed many sets of primers and probe combinations. Through experimental comparison, it was found that the fluorescence signal values ​​of the following primers and probe combinations were stable, and the amplified The amplification effect is good, and the detection ct value of the annealing temperature between 55° C. and 60° C. is the smallest, so the present invention determines that the combination of primers and probes is the best combination for detection of Mycoplasma genitalium by fluorescent quantitative PCR.

[0045] MgPar-F1:C(C / A)CTCCCCACTTTTTTCTAACAT (SEQ ID NO.2)

[0046] MgPar-R1:GTTCA(A / G)ATGTC(A / T)GGTGAA(A / G)AGTTAGG(SEQ ID NO.3)

[0047] MgPar-MGB-P1:FAM-ACCCGGTGGGTGTTTA-BHQ1 (SEQ ID NO.4)

Embodiment 3

[0048] Example 3 Optimization of Experimental Parameters and Method Establishment for Mycoplasma Genitalium Real-time Fluorescent Quantitative PCR Detection

[0049] (1) System annealing temperature optimization: the system annealing temperature was changed from 55°C to 65°C, and the results showed that the system with an annealing temperature of 56-60°C had the best amplification effect.

[0050] (2) Optimization of magnesium ion concentration in the system: MgCl in the system 2 Increase from 0.5 μl to 6 μl successively, each increment is 0.5 μl, and three parallel samples are made for each concentration gradient. Result MgCl 2 The amount added is 3 μl (MgCl 2 The amplification effect of the system is the best when the final concentration is 6mM).

[0051] (3) Optimization of fluorescent quantitative PCR amplification system and amplification conditions

[0052]

[0053] Amplification conditions: pre-denaturation at 95°C for 3-8min, 1 cycle; denaturation at 95°C for 15...

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Abstract

The invention provides a fluorescent PCR primer, probe and detecting kit for detecting mycoplasmas genitalium. According to the fluorescent PCR primer, probe and detecting kit for detecting the mycoplasmas genitalium, by comparing the genomes of all known mycoplasmas genitalium in the prior art, a multicopy repetitive sequence gene with the sequence shown as SEQ ID NO.1 is screened out, and the real-time fluorescent PCR primer and a probe of the multicopy repetitive sequence gene, which separately have nucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, are designed. The detecting kit can reach a mycoplasma genitalium detecting sensitivity as low as 0.5 copies, have no specific amplification and present good specificity for 10 other common mycoplasmas and 7 common mycoplasmas genitalium, and have the advantages of being accurate in detection, high in sensitivity and specificity, simple and rapid, thereby achieving high specimen detecting ability.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a target sequence for detecting mycoplasma genitalium, a fluorescent quantitative PCR primer and a probe, and also relates to a method and a kit for detecting mycoplasma genitalium using the target sequence. Background technique [0002] Mycoplasma genitalium (Mycoplasma genitalium) is an important pathogen that causes human nongonococcal urethritis, and it is also one of the important pathogens of genitourinary tract infections such as prostatitis and pelvic inflammatory disease. In 2015, the U.S. Centers for Disease Control and Prevention (CDC) released the latest version of the "Sexually Transmitted Diseases Treatment Guidelines" (2015 Sexually Transmitted Diseases Treatment Guidelines), which clarified the role of Mycoplasma genitalium in sexually transmitted diseases and attracted more clinical attention. Due to the high nutritional requirements and long culture period of My...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/166C12Q2531/113C12Q2563/107
Inventor 刘云霞高博李睿刘贵明龚杰赵飞
Owner 西安博睿康宁生物医学中心有限公司
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