Primers, probe group, kit and detecting method for duplex detecting of Gardner's bacillus and trichomonas vaginalis
A technology of Gardnerella and Trichomonas vaginalis is applied in the detection field of pathogenic microorganisms, which can solve the problems of less detection of Gardnerella by fluorescence quantitative PCR and joint detection of fluorescent quantitative PCR kit products, etc., and improve the cumbersome operation. situation, ensure high efficiency, and ensure the effect of sensitivity
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Embodiment 1
[0064] Embodiment 1: The present invention detects the design of the primer probe pair of GD / Tv rapidly
[0065] Download the 16S rRNA sequence of Gardnerella, Trichomonas vaginalis ITS sequence, and HBB gene sequence in NCBI, and design primer pairs and probes. The sequences are as follows:
[0066] Table 1 Primer and probe sequences of the present invention
[0067]
Embodiment 2
[0068] Embodiment 2: The establishment of the real-time fluorescence quantitative PCR kit for rapid detection of GD / Tv
[0069] Real-time fluorescence quantitative PCR kit for rapid detection of human GD / Tv, including reaction mixture, sample extraction solution, positive control substance, negative control substance, instructions and box.
[0070] The reaction mixture contains upstream and downstream primers and probes (SEQ ID NO: 1-12), Anstart qPCR Master Mix enzyme mixture, Anstart qPCR Master Mix 5× reaction buffer.
[0071] 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 .
[0072] The primers GD-F, GD-R, Tv-F, Tv-R, HBB-F, and HBB-R have a final concentration of 500 nM.
[0073] The concentration of probes GD-P, Tv-P and HBB-R is 200 nM. ...
Embodiment 3
[0084] Embodiment 3: the rapid detection method of GD / Tv nucleic acid detection kit
[0085] Utilize the kit of embodiment 2 to quickly detect GD / Tv in human vaginal secretions, the specific steps are as follows:
[0086] (1) Nucleic acid extraction: add 1 mL of normal saline to the collection tube of vaginal secretions (10 parts, number 1-10) (use the culture method to determine whether it is infected by GD or Tv, which is the current industry gold standard), shake and wash thoroughly 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, and centrifuge at 13,000 rpm for 5 Minutes, discard the supernatant, add 50 μL of sample extract solution that has been shaken and mixed to the precipitate, vortex the shaker to break up the precipitate (if necessary, gently break up the precipita...
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