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Primer and probe for fast detecting various fungi and identifying strains and application of primer and probe

A fungal and rapid technology, applied in the field of microbial detection, can solve the problems of difficulty in distinguishing the specificity of colonized bacteria and infectious bacteria, laboratory contamination, false positives, etc., and achieve the effect of simple operation, high cost, high throughput, and strong specificity

Active Publication Date: 2017-07-28
HANGZHOU DIAN BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the problem is that there are many factors that cause false positives, such as endotoxin pollution, hemolysis and the use of some antineoplastic drugs can make the detection false positive
However, there are limitations in the diagnosis of fungal infection by traditional PCR technology. The amplified DNA product can easily cause laboratory contamination. A very small amount of contamination can cause false positives and it is difficult to distinguish between colonized bacteria and infectious bacteria, making the specificity low.

Method used

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  • Primer and probe for fast detecting various fungi and identifying strains and application of primer and probe
  • Primer and probe for fast detecting various fungi and identifying strains and application of primer and probe
  • Primer and probe for fast detecting various fungi and identifying strains and application of primer and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Amplification and detection and identification of nucleic acid sequences of common clinical fungi, synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.:

[0043] Primer pairs for amplifying fungal 18sV2 region:

[0044] F1 (SEQ ID NO.1): 5'-GTGGTAATTCTAGAGCTAATACA-3',

[0045] R1 (SEQ ID NO.2): 5'-AATTCTAATACGACTCACTATAGGGGCAGAAATTTGAATGAASCA-3';

[0046] Primer pairs for amplifying fungal 18sV9 region:

[0047] F2 (SEQ ID NO.3): 5'-TTGCTCTTCAACGAGGAAT-3',

[0048] R2 (SEQ ID NO.4): 5'-AATTCTAATACGACTCACTATAGGGACGGAAACCTTGTTACGACT-3';

[0049] Molecular beacons for the detection of fungal 18sV2 region amplicons:

[0050] SEQ ID NO.5: 5'FAM-CGGCGATCGGACTCTTTGATGATTCATAATAACTTTTCGAATCGCCG-DAB3';

[0051] Molecular beacons for the detection of fungal 18sV9 region amplicons:

[0052] SEQ ID NO.6: 5'HEX-CGCGCGTTGTGTTGATTACGTCCCTGCCCTTTGTACGCGCG-DAB3';

[0053] Molecular beacons for the detection of fungal 18sV9 region amplicons:

[0054] SEQ ID NO. 7:...

Embodiment 2

[0055] Embodiment 2: the preparation method of kit.

[0056] (1) Amplification reaction liquid: the reaction liquid contains ph=8.00 1M Tris-Hcl buffer, K + , Mg 2+ , dNTPs (purchased from Thermo scientific), NTPs (purchased from Thermo scientific), primers and molecular beacons (the nucleotide sequence was synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., dissolved in DEPC water), -20°C save;

[0057] (2) 5×Q-solution: the reaction solution contains betaine, BSA, DTT and DMSO, and is stored at -20°C;

[0058] (3) Enzyme reaction solution: the reaction solution contains BSA, reverse transcriptase and T7 RNA polymerase, stored at -20°C;

[0059] (4) Positive control: Extract the RNA of 8 kinds of Candida, 1 kind of Cryptococcus, 1 kind of Aspergillus, 1 kind of Mucormyces and 1 kind of Penicillium respectively, and the concentration of each fungal RNA is finally diluted to 30ng / μL, Store at -20°C;

[0060] (5) Negative control: RNase-free distilled water, stored a...

Embodiment 3

[0061] Embodiment 3: detection method.

[0062] Instrument: Nucleic acid purification 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.

[0063] (1) Preparation of fungal RNA templates: Refer to published literature, use corresponding pretreatment methods for different types of clinical specimens, sputum and other mucus are first hydrated with lysate, and sterile body fluids are directly collected by high-speed centrifugation to remove the upper Clear, add 100ul normal saline to resuspend. Prepare fungal RNA according to the instructions of the fungal nucleic acid extraction kit, and use it as a nucleic acid constant temperature amplification reaction template for future use.

[0064] (2) using the fungal RNA obtained in step (1) as a template, using 2 pairs of specific primers and 3 specific ...

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PUM

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Abstract

The invention discloses a primer, molecular beacon and kit for fast detecting various clinically common fungi and identifying strains and belongs to the technical field of microbiological detection. The sequences of the primer is as shown in SEQ ID No. 1-4, and the sequences of the detecting molecular beacon is as shown in SEQ ID No. 5-7. The primer and the molecular beacon have the advantages that the specific primer and the molecular beacon are designed at conserved sites by comparing the gene sequences of various clinically common fungi, and the primer and the molecular beacon can fast detect and identify 8 clinically common candida, 1 cryptococcus, aspergillus, mucor and penicillium. The kit for detecting various fungi contains the primer and the molecular beacon and can fast and accurately detect various clinically common fungi.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a primer, a molecular beacon and a kit for rapidly detecting a variety of common clinical fungi and identifying bacterial species. Background technique [0002] Mycosis is a disease caused by fungi as pathogens. It includes not only superficial fungal infections that invade the cuticle, hair, and nail plate, but also deep mycoses that invade the dermis, subcutaneous tissue, and internal organs. In recent years, with the extensive use of broad-spectrum antibacterial drugs, glucocorticoids, immunosuppressants and various medical catheters, the development of organ transplantation and long-term treatment of chronic diseases such as tumor treatment, the invasiveness of immunocompromised people and hospitals The incidence of fungal infections has a clear upward trend, mainly caused by Candida, Cryptococcus and Aspergillus, some rare fungi and opportunistic path...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/725C12R1/74C12R1/72C12R1/68C12R1/67C12R1/785C12R1/80C12R1/645
CPCC12Q1/6895
Inventor 任绪义陈书韵赵铃铃
Owner HANGZHOU DIAN BIOTECH CO LTD
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