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Primer probe combination for simultaneously detecting four types of candida and kit thereof

A primer probe and kit technology, applied in the field of microbial detection

Active Publication Date: 2018-05-15
杭州缔蓝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the fluorescent probe method (TaqMan method) is routinely used to detect Candida, and a pair of primers plus a probe determines that its specificity is very high, and there is basically no non-specific amplification, which solves the problem of cross-reaction. Adding a probe to the primer can only detect one species of Candida

Method used

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  • Primer probe combination for simultaneously detecting four types of candida and kit thereof
  • Primer probe combination for simultaneously detecting four types of candida and kit thereof
  • Primer probe combination for simultaneously detecting four types of candida and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] The composition of the kit:

[0116] 1. Primer design

[0117] According to the genomic DNA sequences of the ribosomal RNA of Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis, target sequences that can universally detect these four species and are not homologous to other species were screened. The nucleotide sequence is the same as the nucleotide sequence of the target sequence in the specific embodiment, and will not be repeated here.

[0118] For the target sequences of Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis, specific primers for detecting these four Candida species by fluorescent PCR were designed, and the sequences of the primers were identical to those in the specific embodiment, I won't repeat them here.

[0119] 2. Fluorescent probe design

[0120] For the specific primer sequences and target sequences of the above-mentioned fluorescent PCR, a fluorescent probe specifically binding to the ...

Embodiment 2

[0135] Detection method of the kit

[0136] Before using this kit for detection, it is necessary to extract the DNA of the sample to be tested. The sample to be tested can be sputum, secretions, urine, pleural effusion, ascites, cerebrospinal fluid and other samples that may contain Candida. Extraction and purification of sample DNA A special method is required. The difference from conventional DNA extraction methods is that fungi have relatively thick cell walls. Unlike other common bacteria, viruses and other microorganisms, it is necessary to increase the step of breaking the wall, otherwise the DNA will be difficult to release, resulting in subsequent fluorescent PCR. The failure of the response. For fungal DNA extraction and purification, you can use the fungal-specific extraction kit produced by Hangzhou Dilan Biotechnology Co., Ltd.

[0137] 1. Preparation of PCR reaction tubes (reagent preparation area)

[0138] (1) Determine the number of reaction tubes n (number of...

Embodiment 3

[0163] Performance evaluation of the kit

[0164] 1. Detection limit

[0165] 1) The dose-response curve of this kit and the positive reference substance of Candida albicans

[0166] Candida albicans positive reference substance: different concentrations of Candida albicans cloning plasmid ABc-T.

[0167] Using the kit of Example 1 of the present invention, according to the method of Example 2, the positive reference substance of Candida albicans was detected, and the results are shown in Table 5.

[0168] Table 5 Detection limit of Candida albicans

[0169]

[0170] According to Table 5, it can be concluded that the kit provided by the present application can detect Candida albicans in the range of 1 ng / μl-0.00001 ng / μl.

[0171] 2) The dose-effect curve of this kit and the positive reference substance of Candida tropicalis

[0172] Candida tropicalis positive reference substance: different concentrations of Candida tropicalis cloning plasmid ARc-T.

[0173] Using the...

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Abstract

The invention provides a primer probe combination for simultaneously detecting four types of candida, which belongs to the technical field of microbe detection. The primer probe combination comprisesprimers and a probe, the primer is an upstream primer and a downstream primer, a sequence of the upstream primer is shown as SEQ ID No.1; the sequence of the downstream primer is shown as SEQ ID No.2,and the probe is the sequence labeled with a report fluorescence group and a quenching fluorescence group shown as SEQ ID No.3. The invention provides a pair of primers and a probe which specificallyand simultaneously detect four types of candida such as Candida albicans, Candida tropicalis, candida glabrata and candida parapsilosis.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a combination of primers and probes and a kit for simultaneously detecting four kinds of candida. Background technique [0002] Candida includes about 150 kinds of yeasts that do not produce spores, and is the most common conditional pathogen in fungi. Among them, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida astrocytes and Candida krusei. Among them, Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis accounted for more than 80% of clinical medical samples. Candida often parasitizes on human skin, oral cavity, vagina and intestinal mucosa, etc. When the body's immune function is low or the micro-ecological environment of the normal habitat is out of balance, it is easy to cause candidiasis. Candida can cause superficial or deep skin and mucous membrane infections. Superficial infections mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12R1/725C12R1/74C12R1/72
CPCC12Q1/6895
Inventor 方园任云欧阳云冯莹
Owner 杭州缔蓝生物技术有限公司
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