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Primers for doubly detecting gardnerellavaginalis and blastomyces albicans, probe groups, kit and detecting method

A technology of Candida albicans and Gardnerella, which is applied in the field of probe sets, double detection primers for Candida albicans, and Gardnerella, and can solve the problem of joint detection of fluorescent quantitative PCR kit products and the fluorescence of Gardnerella Quantitative PCR detection is less and other problems, to achieve the effect of ensuring high efficiency, improving cumbersome operation, and reducing the problem of high detection cost

Pending Publication Date: 2019-09-27
中生方政生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] At present, there are many domestic patents for fluorescent quantitative PCR detection of Candida albicans; 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 Albicans and Candida albicans dual detection; while there are very few fluorescent quantitative PCR detections for Gardnerella bacilli, and there is no fluorescent quantitative PCR kit product for GD / CA combined detection

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  • Primers for doubly detecting gardnerellavaginalis and blastomyces albicans, probe groups, kit and detecting method
  • Primers for doubly detecting gardnerellavaginalis and blastomyces albicans, probe groups, kit and detecting method
  • Primers for doubly detecting gardnerellavaginalis and blastomyces albicans, probe groups, kit and detecting method

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Embodiment 1

[0063] Embodiment 1: The present invention detects the design of the primer probe pair of GD / CA rapidly

[0064] Download the 16S rRNA sequence of Gardnerella, Candida albicans ITS sequence, and HBB gene sequence in NCBI, and design primer pairs and probes. The sequences are as follows:

[0065] Table 1 Primer and probe sequences of the present invention

[0066]

Embodiment 2

[0067] Embodiment 2: The establishment of the real-time fluorescent quantitative PCR kit for rapid detection of GD / CA

[0068] Real-time fluorescence quantitative PCR kit for rapid detection of human GD / CA, including reaction mixture, sample extract, positive control, negative control, instructions and box.

[0069] The reaction mixture contains upstream and downstream primers and probes (SEQ ID NO: 1-9), Anstart qPCR Master Mix enzyme mixture, Anstart qPCR Master Mix 5× reaction buffer.

[0070] 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 .

[0071] The primers GD-F, GD-R, CA-F, CA-R, HBB-F, and HBB-R have a final concentration of 500 nM.

[0072] The concentration of probes GD-P, CA-P and HBB-R is 200 nM.

[0073] The GD-P probe fluorescen...

Embodiment 3

[0083] Embodiment 3: the rapid detection method of GD / CA nucleic acid detection kit

[0084] Utilize the kit of embodiment 2 to quickly detect GD / CA in human vaginal secretions, the specific steps are as follows:

[0085] (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 CA, 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 for doubly detecting gardnerellavaginalis and blastomyces albicans, probe groups, a kit and a detecting method. The kit contains specific primer pairs and Taqman fluorescent probes in a GD / CA conserved region. The kit can accurately detect a GD / CA infected sample through real-time fluorescent quantitative PCR, and has the advantages of high sensitivity, good specificity, high repeatability, and convenience and quickness in 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 dual detection of Gardnerella and Candida albicans. 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 treatme...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11C12R1/725C12R1/01
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2563/107C12Q2561/113C12Q2531/113C12Q2537/143
Inventor 邹国宝郭光华蔺皓魏颖颖刘欣欣宋高尚沈江卫吴茜
Owner 中生方政生物技术股份有限公司
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