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Primers and probes for quantitative detection of Candida albicans and their application

A technology for the quantitative detection of Candida albicans, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problem of high false positive rate

Active Publication Date: 2016-02-24
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhejiang University applied for a fluorescent quantitative PCR detection kit for the detection of intestinal Candida albicans (patent number CN200910098555.3), but the SYBRGreen dye method was used. Although this method has high sensitivity, it has a high false positive rate

Method used

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  • Primers and probes for quantitative detection of Candida albicans and their application
  • Primers and probes for quantitative detection of Candida albicans and their application
  • Primers and probes for quantitative detection of Candida albicans and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Preparation of ITS sequence standards:

[0023] In order to establish a real-time fluorescent quantitative PCR method, it is necessary to prepare the external standards required by the method first. The standards should contain highly conserved and specific sequences to ensure high specificity of the reaction. The present invention uses the conserved region of Candida albicans ITS DNA sequence as the target sequence. Mainly use PCR technology to amplify the ITS sequence of Candida albicans, use gene recombination technology to connect it to the plasmid vector pMD18-T, construct the recombinant plasmid pMD18-T-CaITS, and carry out corresponding PCR identification and sequencing identification, and finally After quantification, it is used as a standard for the method to be established, laying the foundation for the next method and evaluation.

[0024] 1. Preparation of template DNA:

[0025] 1. Extract the genomic DNA of Candida albicans (standard strain CMCC98001) and ...

Embodiment 2

[0066] Preliminary establishment of real-time fluorescent quantitative PCR detection method for Candida albicans:

[0067] 1. Design and synthesis of specific primers and probes:

[0068] Taking the conserved fragment of the ITS sequence of Candida albicans selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primerexpress3 software, PrimerPremier5 software and Oligo7 software.

[0069] As the core of the present invention, a group of primers and probe nucleotide sequences for Candida albicans real-time fluorescent PCR detection are as follows:

[0070] Upstream primer: CaITS448F: 5'-CCTAAGCCATTGTCAAAGCGA-3',

[0071] Downstream primer: CaITS331R: 5'-TGCCTGTTTGAGCGTCGTT-3',

[0072] Probe: CaITS426P: 5'-FAM-TCCCGCCTTACCACTACCGTCTTTCAAG-TAMRA-3'.

[0073] The primers and probes amplify the target nucleotide sequence as:

[0074] 5'-TGCCTGTTTGAGCGTCGTTTCTCCCCTCAAACCGCTGGGTTTGGTGTTGAGCAATACGACTTGGGTTTGCTTGAAAGACG...

Embodiment 3

[0087] Evaluation experiment:

[0088] 1. Sensitivity experiment:

[0089] The plasmids prepared above were diluted 10 times to obtain plasmids with a series of concentrations, and the selected concentration was 1.00×10 8 copies / ml, 1.00×10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml, 1.00×10 4 copies / ml, 1.00×10 3 copies / ml, 1.00×10 2 copies / ml etc. as gradient templates. The reaction system is shown in Table 4.

[0090] Table 4

[0091] Element

volume

2×premix

10μl

Primer F (5μM)

1.5μl

Primer R (5 μM)

1.5μl

Probe (5μM)

0.75μl

template

2μl

wxya 2 o

Make up to 20μl

[0092]Reaction conditions: 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40s and collecting fluorescence signals, 40 cycles. According to the fluorescence signal detected by the instrument, the fluorescence curve is obtained by software processing, and the signal of the fluorescence cur...

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Abstract

The invention discloses a primer and a probe for quantitatively detecting Candida Albicans and an application of the primer and the probe. Nucleotide sequences of the primer are shown in sequences 3 and 4 in a sequence table; and a nucleotide sequence of the probe is a sequence 5 in the sequence table. The primer and the probe for quantitatively detecting Candida Albicans and the application of the primer and the probe have the benefits as follows: a group of specific primer and probe sequences for Candida Albicans are provided for qualitative and quantitative detection of Candida Albicans; the Candida Albicans DNA (deoxyribonucleic acid) content in a to-be-detected sample can be accurately quantified by extracting DNA in the to-be-detected sample and combining a real-time fluorescence quantitative PCR (polymerase chain reaction) detection technology; and the primer and the probe can be used for qualitatively and quantitatively analyzing Candida Albicans DNA carried by a patient infected with fungal in clinic and scientific research, have important significance on judgment of Candida Albicans infection, therapeutic effect evaluation and dynamic observation of illness, and simultaneously play an important role in the field of clinical medicine detection.

Description

technical field [0001] The invention relates to a primer and a probe for quantitatively detecting Candida albicans and application thereof. Background technique [0002] Candida albicans (CanidiaAlbicans), also known as Candida albicans, belongs to the subphylum Deuteromycota, Bacillus, Cryptococcales, and Cryptococcalaceae. It exists widely in nature, and also exists in the oral cavity, upper respiratory tract, intestinal tract, and vagina of healthy people. Generally, the total number of normal organisms is small and does not cause disease. Infected tissue and symptoms appear as mycelium. [0003] In recent years, with the abuse of broad-spectrum antibacterial drugs, the increase of cancer radiotherapy and chemotherapy, the clinical application of immunosuppressants and a large number of hormones in organ transplantation, the rate of deep fungal infection has increased significantly, and it has become the main cause of death of patients with the above diseases. Candida a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/06
Inventor 车团结李琳徐进章尤崇革李江西袁皓加
Owner SUZHOU BAIYUAN GENT CO LTD
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