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

Pending Publication Date: 2019-10-18
中生方政生物技术股份有限公司
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Problems solved by technology

[0010] At present, there are less than ten patents for independent detection of Trichomonas vaginalis in China. The patent with the publication number CN102719529B discloses a dual-channel fluorescent PCR detection method and kit for Trichomonas vaginalis and Candida albicans, which is aimed at Trichomonas vaginalis. Double detection of Trichomonas and Candida albicans; while there are very few fluorescent quantitative PCR detections for Gardnerella, and there is no fluorescent quantitative PCR kit product for combined detection of GD / Tv

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  • Primers, probe group, kit and detecting method for duplex detecting of Gardner's bacillus and trichomonas vaginalis
  • Primers, probe group, kit and detecting method for duplex detecting of Gardner's bacillus and trichomonas vaginalis
  • Primers, probe group, kit and detecting method for duplex detecting of Gardner's bacillus and trichomonas vaginalis

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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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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 duplex detecting of Gardner's bacillusand trichomonas vaginalis. The kit comprises GD/TV conserved domain specific primer pairs and Taqman fluorescent probes. Through a real-time fluorescent quantitative PCR, the kit can accurately detect GD/TV infection samples and is high in sensitivity, specificity and repeatability 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 dual detection of Gardnerella and Trichomonas vaginalis. Background technique [0002] Vaginitis, or inflammation of the vagina, is a group of conditions that cause vulvovaginal symptoms such as itching, burning, irritation, and abnormal discharge. Under normal circumstances, there are aerobic bacteria and anaerobic bacteria living in the vagina, forming a normal vaginal flora. Any reason that breaks the ecological balance between the vagina and the flora can also form conditional pathogenic bacteria. Common clinically: bacterial vaginosis (22% to 50% of women with symptoms), candidal vaginitis (17% to 39%), trichomonas vaginitis (4% to 35%), senile vaginitis , Young female vaginitis. The effective identification and diagnosis of the pathogen of vaginitis is very important for the symptomatic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6893C12Q1/686C12Q1/04C12N15/11C12R1/01C12R1/90
CPCC12Q1/6893C12Q1/689C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 蔺皓郭光华邹国宝魏颖颖宋高尚吴茜刘欣欣沈江卫
Owner 中生方政生物技术股份有限公司
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