Primers, probe group, kit and detecting method for multiplex detecting of four types of vagina and urinary tract mycoplasmas

A technology for multiple detection and mycoplasma, which is applied in the field of probe sets and primers for multiple detection of mycoplasma vaginalis, can solve the problems of no joint detection, difficulty in culturing mycoplasma genitalium, inability to distinguish Ureaplasma urealyticum, etc., and achieve good accuracy Effect

Pending Publication Date: 2019-10-18
中生方政生物技术股份有限公司
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  • Claims
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AI Technical Summary

Problems solved by technology

[0008] At present, the detection methods of Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) are mainly isolated culture methods, but the cultivation of Mycoplasma genitalium (MG) is difficult, usually It takes 1-2 hours and the culture method cannot distinguish between Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP)
[0009] The real-time quantitative PCR detection platform has become the most simple and reliable nucleic acid detection method, and it is the most widely used in nucleic acid diagnosis at home and abroad. Domestically, there are kits for the detection of mycoplasma vaginalis alone on the market, but there is no specific kit for MG / MH / Fluorescent quantitative PCR kit product for UU / UP combined detection and differential diagnosis

Method used

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  • Primers, probe group, kit and detecting method for multiplex detecting of four types of vagina and urinary tract mycoplasmas
  • Primers, probe group, kit and detecting method for multiplex detecting of four types of vagina and urinary tract mycoplasmas
  • Primers, probe group, kit and detecting method for multiplex detecting of four types of vagina and urinary tract mycoplasmas

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Experimental program
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Effect test

Embodiment 1

[0069] Embodiment 1: be used for the design of the primer probe pair of rapid detection MG / MH / UU / UP

[0070] Download the mgpC sequence of Mycoplasma genitalium, the 16SrRNA sequence of Mycoplasma hominis / Ureaplasma urealyticum / U. parvum, and the HBB gene sequence in NCBI, and design primer pairs and probes. The sequences are as follows:

[0071] Table 1 Primer and Probe Sequences

[0072]

[0073]

Embodiment 2

[0074] Embodiment 2: The establishment of the real-time fluorescent quantitative PCR kit for rapid detection MG / MH / UU / UP

[0075] Real-time fluorescent quantitative PCR kit for rapid detection of human MG / MH / UU / UP differential diagnosis, including reaction mixture, sample extract, positive control, negative control, instructions and box.

[0076] The reaction mixture contains upstream and downstream primers and probes (SEQ ID NO: 1-13), Anstart qPCR Master Mix enzyme mixture, Anstart qPCR Master Mix 5× reaction buffer.

[0077] Among them, Anstart qPCR Master Mix Enzyme Mixture and Anstart qPCR Master Mix5×Reaction Buffer are provided by Feipeng Biological Co., Ltd. Anstart qPCR Master Mix Enzyme Mixture is diluted 25 times for use, and Anstart qPCR Master Mix5×Reaction Buffer is diluted 5 times for use .

[0078] The primers MG-F, MG-R, MH-F, MH-R, UUUP-F, UUUP-R, HBB-F, and HBB-R have a final concentration of 500 nM.

[0079] The concentration of probes MG-P, MH-P, UU-P, UP-...

Embodiment 3

[0094] Embodiment 3: the rapid detection method of MG / MH / UU / UP nucleic acid detection kit

[0095] Utilize the kit of embodiment 2 to quickly detect MG / MH / UU / UP in human vaginal secretions, the specific steps are as follows:

[0096] (1) Nucleic acid extraction: from female vaginal secretions (5 copies, number 1-5), male urethral secretions (5 copies, number 6-10) (of which 1-6, 2-7, 3-8, 4- 9, 5-10 are husband and wife respectively) (use the culture method to determine whether they are infected by MG, MH, UU or UP, this method is the current industry gold standard) add 1mL of normal saline to the collection tube, shake and wash the cotton swab fully, Then squeeze the cotton swab against the wall and discard it, transfer 500 μL of the liquid to a 1.5 mL centrifuge tube, centrifuge at 13,000 rpm for 5 minutes, discard the supernatant, add 1 mL of normal saline to the precipitate, break up the precipitate, centrifuge at 13,000 rpm for 5 minutes, discard the supernatant Add 50 μ...

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Abstract

The invention relates to the technical field of detection of pathogenic microorganisms, in particular to primers, a probe group, a kit and a detecting method for multiplex detecting of four types of vagina and urinary tract mycoplasmas. The kit comprises MG/MH/UU/UP conserved domain specific primer pairs and Taqman fluorescent probes. Through a real-time fluorescent quantitative PCR, the kit can rapidly and accurately detect MG/MH/UU/UP infection samples and conduct differential diagnosis and is high in specificity and distinction degree and convenient and rapid to use.

Description

technical field [0001] The invention relates to the technical field of detection of pathogenic microorganisms, in particular to primers, a probe set, a kit and a detection method for multiple detection of four kinds of vaginal urogenital mycoplasma. Background technique [0002] Mycoplasma (mycoplasma) is a class of the smallest prokaryotic cell-type microorganisms that have no cell wall, are highly pleomorphic, can pass through bacteria filters, and can be cultured and proliferated in artificial media, with a size of 0.1-0.3 microns. Mycoplasma mainly reproduces by binary fission, and can also reproduce by budding. The branches form filaments and then break into club-shaped particles. Most mycoplasmas reproduce slower than bacteria, the optimum growth temperature is 35°C, and the optimum pH value is 7.8-8.0. Cultivated on solid medium to form typical "poached egg" colonies. Because it can form filamentous and branched shapes, it is called mycoplasma. [0003] Mycoplasma ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/11C12R1/35
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101
Inventor 魏颖颖邹国宝郭光华蔺皓宋高尚刘欣欣沈江卫吴茜
Owner 中生方政生物技术股份有限公司
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