Method for detecting three kinds of urogenital canal mycoplasma by loop-mediated isothermal amplification technology

Abstract

The invention discloses an LAMP detection method for three kinds of urogenital canal infection mycoplasma which are common for human and a dedicated primer and a kit thereof. The method is applied toperforming isothermal amplification on ureaplasma urealyticum (UU), mycoplasma genitalium (MG) and mycoplasma hominis (MH). The LAMP primer is designed according to specific conserved sequences of theUU, the MG and the MH, and each group of the primer includes four piece of oligonucleotide (shown in a table 1, a table 2 and a table 3 in the description). When being applied to the urogenital canalmycoplasma, the LAMP primer present white precipitate when being observed by the naked eyes; in positive reaction, after SYBR GREEN is added, fluorescent green is remarkably enhanced when being observed under an ultraviolet lamp. Detection results of a real-time turbidity meter show that product turbidity can be increased along with prolonging reaction time; after being detected by gel electrophoresis, the LAMP primer is in a trapezoid stripe. The LAMP detection method disclosed by the invention provides a novel technological platform for mycoplasma detection; a sample to be measured can be applied to DNA extracted by commercialized kits or purified DNA and is also suitable for coarse extracted DNA with a boiling method as a representative; the LAMP detection method is suitable for beingpopularized and applied in grassroots units, field monitoring and bedside detection.

Description

technical field [0001] The present invention belongs to the application of a molecular biology detection method represented by isothermal amplification in three common human urogenital infection mycoplasma, that is, the ring-mediated isothermal amplification detection of Ureaplasma urealyticum, Mycoplasma genitalium and Mycoplasma hominis Technology and its special primers, detection methods and kits. Background technique [0002] Mycoplasma (Mycoplasma) is a prokaryotic cell-type microorganism between bacteria and viruses. So far, there are about 20 species of mycoplasma that can cause human infection, among which there are mainly three kinds of mycoplasma that cause human genitourinary tract infection, namely Ureaplasma urealyticum (UU), Mycoplasma genitalium (Mycoplasma Genitalium, MG) and human type Mycoplasma Hominis (MH). Studies have shown that UU, MG and MH are the main pathogens causing nongonococcal urethristis (NGU). In European and American countries, NGU infe...

AI Technical Summary

Problems solved by technology

At present, the gold standard for laboratory diagnosis of mycoplasma is isolation culture, but there are usually two disadvantages in the isolation culture method. One is that the detection time is too long, and it takes at least 3 weeks for typical colonies to grow on solid plates.

Second, it is difficult to isolate and cultivate, some mycoplasma (such as MG) have strict nutritional requirements, the isolation and culture of clinical specimens is not easy to succeed, and it is easy to cause false negative results

[0006] To sum up, the application of LAMP in the detection of Mycoplasma pneumoniae has been reported at home and abroad, but so far, there are no LAMP-specific primers and kits for the detection of UU, MG and MH in the market.

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