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Primer and probe set and kit for multi-detection of gardnerella vaginalis, canidia albicans and trichomonas vaginalis and detection method

A technology of Candida albicans and Gardnerella, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of joint detection of fluorescent quantitative PCR kit products, etc., to avoid pollution and ensure Efficiency and sensitivity, and the effect of improving troublesome operations

Pending Publication Date: 2019-10-18
中生方政生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there are many domestic patents for fluorescent quantitative PCR detection of Candida albicans; for Trichomonas vaginalis, there are less than ten patents for independent detection, and the patent with the publication number CN102719529B discloses the dual The channel fluorescent PCR detection method and its kit are dual detections for Trichomonas vaginalis and Candida albicans; there are very few fluorescent quantitative PCR detections for Gardnerella, and there is no combined detection for GD / CA / Tv Fluorescent quantitative PCR kit products

Method used

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  • Primer and probe set and kit for multi-detection of gardnerella vaginalis, canidia albicans and trichomonas vaginalis and detection method
  • Primer and probe set and kit for multi-detection of gardnerella vaginalis, canidia albicans and trichomonas vaginalis and detection method
  • Primer and probe set and kit for multi-detection of gardnerella vaginalis, canidia albicans and trichomonas vaginalis and detection method

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Effect test

Embodiment 1

[0070] Embodiment 1: The present invention detects the design of the primer probe pair of GD / CA / Tv rapidly

[0071] Download the 16S rRNA sequence of Gardnerella, Candida albicans ITS sequence, Trichomonas vaginalis ITS sequence, and HBB gene sequence in NCBI, and design primer pairs and probes. The sequences are as follows:

[0072] Table 1 Primer and probe sequences of the present invention

[0073]

Embodiment 2

[0074] Embodiment 2: The establishment of the real-time fluorescent quantitative PCR kit for rapid detection of GD / CA / Tv

[0075] Real-time fluorescence quantitative PCR kit for rapid detection of human GD / CA / Tv, 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: 1-12), 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 GD-F, GD-R, CA-F, CA-R, Tv-F, Tv-R, HBB-F, and HBB-R have a final concentration of 500 nM.

[0079] The concentration of probes GD-P, CA-P, Tv-P and HBB-R is 200 nM.

[0080] ...

Embodiment 3

[0092] Embodiment 3: the rapid detection method of GD / CA / Tv nucleic acid detection kit

[0093] Utilize the kit of embodiment 2 to quickly detect GD / CA / Tv in human vaginal secretions, the specific steps are as follows:

[0094] (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, CA or Tv, this method is the current industry gold standard), and fully Shake and wash the cotton swab, 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 13000 rpm for 5 minutes, discard the supernatant, add 1 mL of normal saline to the precipitate, break up the precipitate, 13000 rpm Centrifuge for 5 minutes, discard the supernatant, add 50 μL of sample extract solution that has been shaken and mixed to the pellet, shake on a vortex shaker to break up the precipitate (if necessary,...

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Abstract

The invention relates to the technical field of detection of pathogenic microorganisms, in particular to a primer and probe set and kit for multi-detection of gardnerella vaginalis, canidia albicans and trichomonas vaginalis and a detection method. The kit comprises specific primer pairs and Taqman fluorescence probes for conservative areas of GD / CA / TV, and the kit can accurately detect samples infected with GD / CA / TV through real-time fluorescence quantification PCR and is high in sensitivity, good in specificity, high in repeatability and convenient and quick to use.

Description

technical field [0001] The invention relates to the technical field of detection of pathogenic microorganisms, in particular to primers, probe sets, kits and detection methods for multiple detection of Gardnerella, Candida albicans 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/689C12Q1/6893C12Q1/6851C12Q1/06C12N15/11C12R1/725C12R1/90C12R1/01
CPCC12Q1/6895C12Q1/689C12Q1/6893C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2545/101C12Q2537/143C12Q2563/107
Inventor 郭光华魏颖颖蔺皓邹国宝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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