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Deep fungal infection detection kit and detection method thereof

A technology for infection detection and deep fungus, applied in the field of biomedical diagnosis, can solve the problems of high false positive detection, false negative detection, low detection rate, etc., and achieve high sensitivity, high throughput, and easy operation

Inactive Publication Date: 2019-12-31
南京东万先卓生物科技研发有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although direct microscopic examination is fast and simple, the detection rate is low, and false negatives may occur. False negatives cannot rule out the possibility of fungal infection. It usually needs to be identified jointly with microbial culture methods, but the culture method takes as long as 5-7 days. Individual fungal cultures even as long as 20 days
Although the G test and GM test have strong specificity, they have high false positive detection

Method used

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  • Deep fungal infection detection kit and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: reagent composition

[0027] (1) DNA extraction reagents

[0028] Solution I, Solution II, Solution III, Solution IV. Solution Ⅰ contains 20 mM / L-100 mM / L Tris, 10 mM / L-50 mM / L EDTA, 4-8M / L guanidine hydrochloride, pH 4.0-8.0; solution Ⅱ is β-mercaptoethanol; solution Ⅲ is 75 %-95% ethanol; solution IV is ddH 2 O, pH 7.0-9.0.

[0029] (2) PCR amplification components

[0030] 3 μl 10×Buffer, 0.6 μl 4×dNTP, 1.8 μl CA, 1.8 μl CN, 1.8 μl AF, 0.2 μl Taq DNA polymerase, 1.8 μl MgCl 2 , 3μl sample template and RNase-free water;

Embodiment 2

[0031] Embodiment 2: application embodiment 1 kit is detected

[0032] (1) Extraction of fungal DNA: Taking the cultivation of Candida albicans as an example, the specific steps are as follows:

[0033] (1a) Take culture to 1-9×10 7 CFU / ml bacterial suspension 100-500μl, add 10mg / ml-50mg / ml helicase, dissolve, add 0.4%-2.5% solution II, keep warm at 37°C for 10-60min;

[0034] (1b) Add 100-500 μl solution I, mix well, add to the spin column, and centrifuge at 8000r for 1min;

[0035] (1c) Add 500 μl solution III, centrifuge at 10000r for 2 minutes, open the lid and place at room temperature to completely evaporate the residual solution;

[0036] (1d) Add 20-50 μl solution IV, and centrifuge at 10000r for 1min.

[0037] (2) Use the DNA extracted in (1) as a template, and use primers for PCR amplification;

[0038] The amplification system includes: 3 μl 10×Buffer, 0.6 μl 4×dNTP, 1.8 μl CA, 1.8 μl CN, 1.8 μl AF, 0.2 μl Taq DNA polymerase, 1.8 μl MgCl 2 , 3μl sample templat...

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Abstract

The present invention discloses a fungal DNA extraction method and a two-step PCR amplification technology for detecting clinical fungal infections. The method comprises simple DNA extraction method,design of specific primers for candida albicans, cryptococcus neoformans, and aspergillus fumigatus, and establishment of a detection method for simultaneously detecting the above three fungi. The method is sensitive and specific, and has important significance for clinical diagnosis of three fungal infections.

Description

technical field [0001] The invention belongs to the field of biomedical diagnosis, and in particular relates to a deep fungal infection detection kit. Background technique [0002] In recent years, due to the aging population, organ transplantation, tumor radiotherapy and chemotherapy, hematopoietic stem cell transplantation, the application of ultra-wide antibiotics and various catheter interventional treatments, the incidence of fungal infection has increased year by year. According to the site of infection, it can be divided into superficial infection and deep infection. Superficial infection includes skin, mucous membrane, nails, hair, subcutaneous tissue, etc., and deep infection includes lung, blood, gastrointestinal tract and other organ and tissue infections. The current effective treatment for fungal infections is still the use of antibiotics, so accurate and timely diagnosis of the disease is very important. [0003] Currently, clinical methods for diagnosing fung...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/725C12R1/68C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 乐龙尹鸿萍刘永军
Owner 南京东万先卓生物科技研发有限公司
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