LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida

A technology for Candida glabrata and Candida spp. is applied in the biological field to achieve the effects of improving efficiency, being suitable for industrial promotion and use, and having highly specific test results.

Active Publication Date: 2021-04-20
中迅优检生物科技(江苏)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the products used for the detection of candida that have obtained the CFDA medical device registration certificate in my country mainly use the traditional fluorescent PCR detection method, which still has room for improvement in detection sensitivity and detection specificity

Method used

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  • LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida
  • LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida
  • LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Design and screening of embodiment 1 primer / probe system

[0073] 1. Preparation of nucleic acid amplification reagents

[0074] DNA polymerase includes: 1 μL of 5U / μL DNA polymerase;

[0075] The buffer included: 2 μL of 1M KCl; 0.5 μL of 0.4M MgCl2; 1 μL of 10% Tween 20; 4 μL of 25 mM dNTPs; 1.5 μL of 0.2M Tris-Hcl (pH=8.0).

[0076] 2. Design of PCR amplification primers and Taqman probes

[0077] In the present invention, firstly, according to the standard sequences of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei included in GenBank, the determined intra-species conservative and inter-species specific sequence segments are screened, specifically as Shown in SEQID NO.1-5; and then artificially design PCR amplification primers and Taqman probe sequences according to the determined sequence segments, and compare and analyze them. All primer sequences were synthesized by China Shenggong Company.

[0078] Specificall...

Embodiment 2

[0100] Performance verification of embodiment 2PCR amplification primers and LNA-Taqman probes

[0101] 1. Specificity verification

[0102] Utilize the Cal group primer probe combination, reaction system and detection method that embodiment 1 establishes, by carrying out LNA-Taqman multiplex fluorescent PCR detection to 1 strain Candida albicans genomic DNA and 4 kinds of other Candida species; Utilize embodiment 1 to determine Cgla group primer probe combination, reaction system and detection method, by carrying out LNA-Taqman multiple fluorescent PCR detection to 1 strain Candida glabrata genomic DNA and 4 kinds of other Candida species; Utilize the Ctro group primer probe obtained in embodiment 1 Combination, reaction system and detection method, by carrying out LNA-Taqman multiplex fluorescent PCR detection to 1 strain Candida tropicalis genomic DNA and 4 kinds of other Candida species; Utilize the Cpa group primer probe combination that obtains in embodiment 1, reaction ...

Embodiment 3

[0112] Embodiment 3 Clinical test book detection and comparison of different detection methods

[0113] 1. Collection of clinical samples and preparation of nucleic acid genome DNA of clinical samples

[0114] The genital secretion samples (i.e., vaginal swabs) of 993 women at 16 weeks of pregnancy were collected and provided by Beijing Obstetrics and Gynecology Hospital. Extraction of DNA from clinical samples: Genomic DNA extraction from clinical samples was performed using Tiangen’s Universal Genomic DNA Extraction Kit (catalogue number: DP705) by magnetic bead method. Refer to step H in the manual for details. The extracted genomic DNA of clinical samples was measured with a NanoDrop 2000C ultra-micro spectrophotometer for the concentration and purity of Candida genomic DNA, and stored in a -20°C refrigerator for later use.

[0115] 2. Detection and identification of clinical samples by LNA-TaqMan multiplex fluorescent PCR

[0116] Preparation and amplification conditio...

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Abstract

The invention relates to the technical field of biotechnology, in particular to LNA-Taqman-multiplex fluorescence PCR and application thereof in candida detection. According to the present invention, the system of specific amplification primer and LNA-Taqman probe can be used for rapid amplification detection of common candida in female genital tract, and compared with the traditional detection method, the detection efficiency, the sensitivity, the specificity and the multiple detection rate are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an LNA-Taqman-multiple fluorescent PCR technology and its application in the rapid detection of candida in female reproductive tract secretions. Background technique [0002] Vaginal yeast infections, also known as candidal vaginitis and vaginal thrush, are caused by an overgrowth of the fungus Candida. These yeasts are usually found in small amounts in the vagina. The most common symptom is vaginal itching. Other symptoms include burning when urinating and thick vaginal discharge. Symptoms often worsen before women go into labor. About 75 percent of women have at least one type of vaginal yeast infection at some point in their lives, and nearly half have at least two. About 5% of people get three or more infections in a year. It is the second most common cause of vaginal inflammation after bacterial vaginosis. [0003] Although Candida albicans is the most common yeast associate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/725C12R1/74C12R1/72
Inventor 殷耀宏王校乔毅
Owner 中迅优检生物科技(江苏)有限公司
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