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Kit used for detecting candida tropicalis in intestinal tract by fluorescence quantitative PCR method

A technology for the quantification of Candida tropicalis and fluorescence, which is applied in the directions of fluorescence/phosphorescence, microbiological-based methods, and microbiological determination/inspection. It can solve the problems of long diagnostic cycle and low detection sensitivity, and achieve simple operation, high sensitivity and specificity good sex effect

Inactive Publication Date: 2009-10-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complex structure of the human intestinal microbiome, there are about 10 14 There are different microbial organisms, while the proportion of fungi is low, less than 1%. The growth of some fungal strains also requires complex culture conditions, and the isolation culture method can only detect a small part of microorganisms with suitable conditions, and the detection sensitivity Low and long diagnostic cycle

Method used

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  • Kit used for detecting candida tropicalis in intestinal tract by fluorescence quantitative PCR method
  • Kit used for detecting candida tropicalis in intestinal tract by fluorescence quantitative PCR method
  • Kit used for detecting candida tropicalis in intestinal tract by fluorescence quantitative PCR method

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specific Embodiment 1

[0033] The test kit in this embodiment includes:

[0034] 10×Taqman PCR buffer of 1 / 10 of the total reaction volume;

[0035] Each 0.15 μmol / L upstream and downstream primers;

[0036] Each 200μmol / L dATP, dTTP, dGTP, dCTP;

[0037] 2.0mmol / L MgCl 2 ;

[0038] 0.05U / μl HOTSTART Taq DNA polymerase;

[0039] 0.02×SYBR green I fluorescent dye;

[0040] Standard positive template, its concentration is serially diluted according to the requirements of quantitative standard curve preparation;

[0041] The balance is sterile double distilled water.

specific Embodiment 2

[0042] The kit components are as follows:

[0043] 10×Taqman PCR buffer of 1 / 10 of the total reaction volume;

[0044] Each 1.0 μmol / L upstream and downstream primers;

[0045] Each 200μmol / L dATP, dTTP, dGTP, dCTP;

[0046] 3.5mmol / L MgCl 2 ;

[0047] 0.05U / μl HOTSTART Taq DNA polymerase;

[0048] 0.02×SYBR green I fluorescent dye;

[0049] Standard positive template, its concentration is serially diluted according to the requirements of quantitative standard curve preparation;

[0050] The balance is sterile double distilled water.

specific Embodiment 3

[0051] The kit components are as follows:

[0052] 10×Taqman PCR buffer of 1 / 10 of the total reaction volume;

[0053] Each 0.3 μmol / L upstream and downstream primers;

[0054] Each 200μmol / L dATP, dTTP, dGTP, dCTP;

[0055] 2.5mmol / L MgCl 2 ;

[0056] 0.05U / μl HOTSTART Taq DNA polymerase;

[0057] 0.02×SYBR green I fluorescent dye;

[0058] Standard positive template, its concentration is serially diluted according to the requirements of quantitative standard curve preparation;

[0059] The balance is sterile double distilled water.

[0060] The working principle of the kit of the present invention is introduced:

[0061] In the PCR reaction system, adding excess SYBR fluorescent dye, SYBR fluorescent dye specifically incorporated into DNA double strands, emits a fluorescent signal, and the SYBR dye molecules that are not incorporated into the strand will not emit any fluorescent signal, thus ensuring the fluorescent signal The increase of is fully synchronized with the...

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Abstract

The invention relates to a kit, and aims at providing a kit used for detecting candida tropicalis in the intestinal tract by a fluorescence quantitative PCR method. The kit comprises a standard positive template and two primers of a specificity amplification candida tropicalis ITS zone used in the PCR amplification process, wherein the gene of a coded amplification product has the nucleotide sequence shown by SEQ ID NO: 3. The operation is simple and convenient and fast; the specificity is good, and the sensitivity is high; the detection based on nucleotide is not limited by culture conditions; the quantitative detection can truly reflect the situations of candida tropicalis permanent planting and infection in the intestinal tract; and high-flux sample detection can be implemented simultaneously. The kit can carry out fast and quantitative detection to the candida tropicalis in the intestinal tract, can replace the traditional diagnostic method of isolated culture which is used for a long time, and is suitable for being widely popularized and applied in clinical laboratory.

Description

technical field [0001] The invention relates to a kit, in particular to a kit for detecting intestinal Candida tropicalis by fluorescent quantitative PCR method. Background technique [0002] A rich and diverse fungal community exists in the normal human gut. Many studies have been carried out on human intestinal fungi using traditional isolation and culture methods (Khatib R, Riederer KM, Ramanathan J, et al. Faecalfungal flora in healthy volunteers and patients. Mycoses, 2001, 44: 151-6.), The method diagnoses the fungal phenotype, observes the growth and reproduction of the colony on the solid medium, identifies the fungus according to the shape, size, color, viscosity and other structural characteristics of the colony, and combines different physiological, biochemical and heat resistance tests, etc. Make a morphological diagnosis. Some pathogenic fungi can be accurately diagnosed by the isolation and culture method. However, in some special cases, such as the colony mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/74
Inventor 李兰娟陈瑜郭仁勇鲁海峰陈珍晶
Owner ZHEJIANG UNIV
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