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Rapid diagnosis kit for detecting three or less invasive candida and application method

A technology for rapid diagnosis and application methods, applied in the directions of microorganism-based methods, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of inappropriate use of tissue biopsy methods, low positive rate, limited application, etc., to reduce mortality. and sequelae, high detection sensitivity, fast detection effect

Inactive Publication Date: 2009-11-25
THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT) +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fungal culture is affected by factors such as sampling time, sampling location, sample factors, and culture conditions. The positive rate has always been low, and it is time-consuming and laborious. Routine culture takes 3 days and cannot play a role in guiding clinical early targeted treatment.
Although the serological method is widely welcomed by laboratories due to its simple operation process, the application range of this method is narrow, and there are cross-antigens, which are prone to false positives or false negatives, and the species cannot be identified, which cannot meet the needs of diagnosis
Histopathological identification method is a method for the diagnosis of clinical invasive candidiasis, but because it is an invasive examination, it is difficult for patients to accept, and patients with severe invasive fungal infection are often accompanied by thrombocytopenia and poor coagulation function, so it is generally not suitable tissue biopsy method
The method has a low positive rate
Therefore, the aforementioned laboratory diagnostic methods limit their application in clinical diagnosis.

Method used

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  • Rapid diagnosis kit for detecting three or less invasive candida and application method
  • Rapid diagnosis kit for detecting three or less invasive candida and application method
  • Rapid diagnosis kit for detecting three or less invasive candida and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] This embodiment 1 constitutes a rapid diagnostic kit for detecting three kinds of Candida.

[0032] It includes a box body and a fluorescent quantitative PCR reaction solution arranged in the box body, positive quality control products and negative quality control products of three kinds of Candida; the fluorescent quantitative PCR reaction solution contains specific primers and fluorescent lights corresponding to the three kinds of Candida Probe; the positive quality control product of the invasive Candida is the DNA sample obtained by the standard strain of Candida albicans, Candida krusei and Candida glabrata through "boiling method", and the negative quality control of the invasive Candida The product is sterilized triple distilled water.

[0033] The preparation method of described three kinds of substances is as follows:

[0034] 1) Preparation of fluorescent quantitative PCR reaction solution: Brilliant QPCR Master mix II 12.5μl, universal primer (0.1mmol / l)

...

specific Embodiment 2

[0052] The characteristics of this specific embodiment 2 are: the three probes are as follows:

[0053]

[0054] 2) Candida albicans, Candida krusei and Candida tropicalis positive quality control products: DNA samples obtained from three kinds of Candida species by "boiling method", stored at -20°C.

[0055] Shown in conjunction with table 2, table 3, use kit of the present invention to amplify 13 kinds of 94 clinically common pathogenic fungi, 5 kinds of bacteria and human whole blood cell DNA, Candida albicans, Candida krusei and Candida tropicalis The Ct values ​​were all ≤ 35.0, and the identification results were positive, while other Ct values ​​such as Candida, bacteria, Aspergillus, Cryptococcus neoformans, and human genomic DNA had no readings, and the results were negative.

[0056] The results of the data in Table 2 and Table 3 fully prove that the kit of the present invention has good specificity.

[0057] All the other are with specific embodiment 1.

specific Embodiment 3

[0058] Dilute any two or three of the suspensions of Candida albicans, Candida krusei, Candida glabrata, and Candida tropicalis by 10 times: 5×10 8 , 5×10 7 , 5×10 6 , 5×10 5 , 5×10 4 , 5×10 3 , 5×10 2 , 5×10 1 CFU / ml. Fluorescent quantitative PCR amplification was carried out after the DNA was extracted according to the above method, and the result was 5×10 3 The "S" curve can still be observed in CFU / ml, indicating that the fluorescence quantitative PCR method can detect at least 5×10 3 CFU / ml of the above four kinds of Candida, the kit of the present invention has strong sensitivity.

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Abstract

The invention relates to a rapid diagnosis kit for detecting three or less invasive candida, which comprises a kit, and is characterized in that: 1) a fluorescent quantitative PCR reaction liquid, 1-3 invasive candida positive quality control materials and negative quality control materials are provided in a kit; 2) a fluorescent quantitative PCR reaction liquid contains a specific primer and a fluorescent probe; 3) the positive quality control materials are DNA samples obtained by boiling of one, optional two or three combination of the standard strains of the candida; the negative quality control materials are sterilized distilled water for three times. The application method comprises following steps: extracting DNA from the sample to be detected; 2) adding the DNA and the positive quality control materials with the equal amount to the PCR reaction system containing the fluorescent quantitative reaction liquid; 3) detecting with the fluorescent quantitative PCR detector, judging the detection result based on the Ct value of the reaction result. The invention can synchronously detect 1-3 invasive candida, provides the evidence for the early pathogen diagnosis of the clinical invasive candidiasis and reduces death rate and sequelae.

Description

technical field [0001] The invention relates to a rapid diagnostic kit and an application method thereof, in particular to a fluorescent quantitative PCR rapid diagnostic kit for detecting three / less than three types of invasive candida and an application method thereof. It belongs to the technical field of fungal nucleic acid detection and is suitable for the rapid detection of Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis and other invasive candida in clinical and scientific research. Background technique [0002] In recent years, with the extensive development of organ transplantation and bone marrow transplantation, the increase of AIDS and cancer patients, and the extensive application of corticosteroids, broad-spectrum antibiotics, and immunosuppressants, the incidence of invasive candida infection has increased significantly. A prospective study in the United States showed that fungal infections accounted for 9.5% of nosocomial bloodstream inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/645
Inventor 黎毅敏杨淳黄红川刘晓青何为群袁锦屏杨灵邱桂霞张彦峰
Owner THE FIRST AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV (GUANGZHOU RESPIRATORY CENT)
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