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
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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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