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Kit applying the molecule beacon-melting curve technology for identifying clinic common fungi and application thereof

A molecular beacon and technical identification technology, which is applied in the field of kits for rapid identification of common clinical fungi, can solve problems such as product aerosol pollution, achieve fast, reliable and accurate identification, improve stability, and avoid false positive pollution.

Pending Publication Date: 2019-08-23
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, any detection system that relies on PCR amplification cannot avoid a series of problems such as false positives due to slight contamination due to its high sensitivity, and aerosol contamination of the amplified product.
[0007] To sum up, there is still no ideal rapid identification product for fungi, which not only meets the needs of fast and efficient, easy operation, cost-effective, high sensitivity, and high throughput; but also has stable results, good repeatability, and simple data interpretation schemes. , can realize the characteristics of automation

Method used

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  • Kit applying the molecule beacon-melting curve technology for identifying clinic common fungi and application thereof
  • Kit applying the molecule beacon-melting curve technology for identifying clinic common fungi and application thereof
  • Kit applying the molecule beacon-melting curve technology for identifying clinic common fungi and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Molecular beacon oligonucleotide sequences used to identify common clinical fungi, synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.:

[0046] Fungal ribosomal RNA V9 molecular beacon SMB1 (SEQ ID NO.1): 5'-HEX-CCGCGGCCTCTAAATGACCAAGTTTGACCAGCTTCTCGGCTCCAGAATCCGCGG-DAB-3';

[0047] Fungal ribosomal RNA V9 molecular beacon SMB2 (SEQ ID NO.2): 5'-CY5-CGGCGCCTAAGCCAATCCGGAGGCCTCACTAAGCCATTCAATCGGTAGCGCCG-DAB-3';

[0048] Fungal ribosomal RNA V4 molecular beacon SMB3 (SEQ ID NO.3): 5'-FAM-CCGCGGCATCAGAAAGATTGACCGGCCAACCAAGCCCAAAGTTCAACTACCCGCGG-DAB-3'.

Embodiment 2

[0049] Embodiment 2: the preparation method of kit.

[0050] (1) HQ buffer: the buffer contains 40mM Tris-Acetate pH7.8-8.0, 100mM potassium acetate, 30mM magnesium acetate, 0.5M betaine, 1.3mM dithiothreitol (DTT), 11μg / mL bovine Serum albumin (BSA), 1.3% (v / v) dimethyl sulfoxide (DMSO) and DEPC water (except that DEPC water was purchased from Shanghai Sangon Biological Co., Ltd., the rest were purchased from Sigma-Aldrich, USA) , stored at -20°C;

[0051] (2) Molecular beacon mixture: The nucleotide sequences shown in SEQ ID NO.1-3 were synthesized by Yingweijieji (Shanghai) Trading Co., Ltd. Dissolve the powder in double distilled water and mix in a tube. The dilution concentration of molecular beacon SMB1 is 7.5 μmol / L, the dilution concentration of molecular beacon SMB2 is 7.5 μmol / L, the dilution concentration of molecular beacon SMB3 is 10 μmol / L, and store at -20 °C;

[0052] (3) Positive control substance: Inoculate Candida albicans ATCC10231 and Candida glabrata A...

Embodiment 3

[0054] Embodiment 3: detection method.

[0055] Instrument: Roche 480 fluorescent quantitative PCR detector, BECKMAN 22R desktop micro-refrigerated centrifuge, Eppendorf 5810R desktop refrigerated centrifuge, Taicang Hualida Laboratory Equipment Company WH-866 vortex oscillator.

[0056] (1) Preparation of fungal RNA template (sample preparation area): the kit of the present invention does not provide a nucleic acid extraction reagent, and the user can select a fungal RNA extraction kit or a fungal total nucleic acid extraction kit to extract fungal nucleic acid containing 18s ribosomal RNA;

[0057] (2) Preparation of reaction reagents (reagent preparation area): Take out the HQ buffer and molecular beacon mixture from -20°C, and after melting at room temperature, prepare the reaction solution X μL according to the number of samples:

[0058] X=(8 μL HQ buffer solution+4.5 μL molecular beacon mixture)×(N samples to be tested+1 negative control substance+2 positive control...

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Abstract

The invention discloses a molecule beacon and kit for rapidly detecting various clinic common pathogen fungi, and belongs to the technical field of microorganism detection. The sequence of the molecule beacon is shown in SEQ ID NO.1-3, by comparing 18s ribosome RNA sequences of various clinic common fungi in an NCBI gene sequence base, the specific molecule beacon is designed for key loci of V4 and V9 variable regions, 17 clinic common fungi of candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, candida krusei, candida lusitaniae, candida guilliermondii, candida koumiss, candida rugosa, brewer's yeast, marine yeast, rhodotorula glutinis, cryptococcus neoformans, cryptococcus laurentii, Cryptococcus humicola, Trichosporon asahii and aspergillus fumigatus can be rapidly detected and identified. The invention further discloses a kit comprising the molecule beacon for identifying various fungi, and the kit can rapidly and accurately identify various clinic commonfungi.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a kit for rapidly identifying common clinical fungi; and also relates to a molecular beacon in the kit that specifically binds to fungal 18s ribosomal RNA sequences. Background technique [0002] Fungal infections can be divided into superficial mycosis and deep mycosis according to the different lesion sites. In recent years, with the development and application of broad-spectrum antibiotics, antineoplastic drugs and immunosuppressants, the widespread development of interventional therapy, organ transplantation, bone marrow transplantation and catheter intubation, the prevalence of immune disorders and AIDS, hospital deep fungal The incidence of infection is rising sharply, and it has become the fourth most common pathogen causing bloodstream infection. Deep fungal infection has two characteristics. One is that the patient’s clinical symptoms and signs a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6816C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6816C12Q2525/301C12Q2527/107C12Q2545/113Y02A50/30
Inventor 任绪义陈书韵
Owner HANGZHOU D A GENETIC ENG