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Primers and kit for fluorescent PCR detection of nine dermatophytes

A dermatophyte and kit technology, which is applied in the field of primers and kits for fluorescent PCR detection, can solve the problems of insignificant clinical early diagnosis, long culture period, low sensitivity of direct microscopy and the like

Active Publication Date: 2020-05-29
杭州缔蓝生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sensitivity and specificity of the direct microscopic examination method are low, and it is difficult to identify the species level; the positive rate of the fungal culture method is very low, generally not more than 20%, and the culture period is long, generally 2 to 3 weeks. Diagnostically insignificant; immunological testing is difficult to establish because dermatophytosis is only a superficial infection

Method used

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  • Primers and kit for fluorescent PCR detection of nine dermatophytes
  • Primers and kit for fluorescent PCR detection of nine dermatophytes
  • Primers and kit for fluorescent PCR detection of nine dermatophytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Composition of the kit

[0062] 1. Primer design

[0063] Ribose based on Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Trichophyton trichophyton, Trichophyton schlendens, Epidermophyton flocculum, Trichophyton verrucous, Microsporum canis and Microsporum gypsumi Genomic DNA sequence of body RNA, screen the target sequence that can universally detect these 9 species of dermatophyte and is different from other species, design specific primers for fluorescent PCR detection, entrust Shanghai Jierui Biotechnology Co., Ltd. to synthesize.

[0064] The nucleotide sequences of the primer sequences and target sequences are as follows:

[0065] The base sequence of the specific primer is:

[0066] Upstream primer: 5'-GTAGGTGAACCTGCGGAAG-3' (SEQ ID NO: 1)

[0067] Downstream primer: 5'-ACCGGGTAAGGTAGACAAG-3' (SEQ ID NO:2)

[0068] The target gene fragments of 9 kinds of dermatophytes can be multiplexed simultaneously in the same tube by using the a...

Embodiment 2

[0112] Detection method of the kit

[0113] Before using this kit for detection, it is necessary to extract the DNA of the sample to be tested. The fungal nucleic acid extraction or purification kit produced by Hangzhou Dilan Biotechnology Co., Ltd. can be used for DNA extraction.

[0114] 1. Preparation of PCR reaction tubes (reagent preparation area)

[0115] (1) Determine the number of reaction tubes n (number of samples + negative control + positive control); take out sterilized purified water (prepared by the user) and PCR reaction solution; take out other components in the kit, put them on ice or Thaw at room temperature. All kit components require brief centrifugation before use. Each reaction system is shown in Table 2:

[0116] Table 2. Sample loading conditions

[0117] PCR reaction solution Primer Sterilized purified water Sample / Control total capacity 10 μL 0.8μL 7.2μL 2μL 20 μL

[0118] Calculate the amount of each of the ab...

Embodiment 3

[0146] Identification of strains

[0147] In order to ensure the accuracy of the kit detection, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schlennium, Trichophyton trichophyton and Trichophyton violaceum, Epidermophyton flocculus, Trichophyton verrucous, Microsporum canis and Microsporum gypsumi were sequenced and verified, and the sequencing was performed on the ITS region and the D1 / D2 region of 28S, and the sequencing primers for the ITS region (ITS-1:

[0148] 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 14); ITS-4:

[0149] 5'-TCCTCCGCTTATTGATATGC-3'(SEQ ID NO:16)), 28S D1 / D2 region sequencing primer (NL-1:5'-GCATATCAATAAGCGGAGGAAAAG-3'(SEQ ID NO:17); NL-4:5' - GGTCCGTGTTTCAAGACGG-3' (SEQ ID NO: 18)).

[0150] The 9 kinds of dermatophytes detected by this kit were all sequenced by the above method to confirm that their respective strains were correct, and then used for the strain verification experiment of this kit.

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Abstract

The invention relates to primers and kit for fluorescent PCR detection of nine dermatophytes, and belongs to the technical field of in-vitro molecular detection of pathogenic microorganisms. The primers include an upstream primer with a nucleotide sequence which is shown in SEQ ID No: 1 and a downstream primer with a nucleotide sequence which is shown in SEQ ID No: 2, wherein the nine dermatophytes include Trichophyton rubrum, Trichophyton mentagrophyte, Trichophyton violaceum, Trichophyton schoenleinii, Epidermophyton floccosum, Trichophyta verruca, Microsporum canis and Microsporum gypsum. The detection is fast, sensitive, specific and repeatable through the primers, and the primers can be applied to early, non-invasive and accurate detection of clinical patients, so that assistance of clinical and effective antifungal therapy is achieved.

Description

technical field [0001] The invention relates to the technical field of in vitro molecular detection of pathogenic microorganisms, in particular to primers and a kit for fluorescent PCR detection of nine kinds of dermatophytes. Background technique [0002] Dermatophytes are superficial fungi, and dermatophytes are mainly composed of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Trichophyton trichophyton, Trichophyton xulan, Epidermophyton flocculus, Trichophyton verrucous , Microsporum canis, Microsporum gypsum, Microsporum rust, etc. caused by more than 30 kinds of dermatophytes, clinically manifested as tinea capitis, onychomycosis, tinea manuum and tinea corporis, etc., is the most common in the crowd One of the skin infections. Dermatophyte infection is generally transmitted through contact, and can infect itself and others. The fungus not only invades the epidermis, but also invades the dermis and even the subcutaneous tissue. Because the fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2600/16C12Q2563/107C12Q2537/143
Inventor 李萌王建定郑方伟孙彩娟方园柯金枝张玉玉
Owner 杭州缔蓝生物技术有限公司
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