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Reagent set, kit and detection method for detecting fungal infection

A fungal infection and reagent group technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as low reliability, time-consuming, and inability to test subjects, and achieve The effect of improving reliability and accuracy, improving specificity and sensitivity, and reducing the risk of cross-contamination

Active Publication Date: 2019-02-01
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, molecular biology methods are expensive, complicated to operate, not standardized, and have not been used in clinical practice on a large scale.
At present, most of the fungal detections on the market are culture methods or fungal (1-3)-β-D-glucan detection kits, and there are no mature kits for other related detection methods. For the culture method detection kits, the results The accuracy rate is high, but it takes a long time, and it is impossible to quickly detect the subjects; the fungal (1-3)-β-D-glucan detection kit detects the cell wall components of fungi, and this method can be applied to IFD early detection of infection, but the detection is prone to false positives in the following situations (1) using cellulose membrane for hemodialysis, specimens or patients exposed to gauze or other materials containing dextran; (2) intravenous infusion of immunoglobulin, Albumin, coagulation factors, or blood products; (3) streptococcemia; (4) operator contamination during specimen handling
In addition, the use of polysaccharide anticancer drugs, mucosal damage caused by radiotherapy and chemotherapy may also cause dextran in food or colonized Candida to enter the blood through the gastrointestinal tract, which may also cause false positives. Eating fungi, such as mushrooms and other foods can lead to false positives
Therefore, the error of the test result is large, the false positive rate is high, and the reliability is low.

Method used

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  • Reagent set, kit and detection method for detecting fungal infection
  • Reagent set, kit and detection method for detecting fungal infection
  • Reagent set, kit and detection method for detecting fungal infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The kit for detecting fungal infection provided in this embodiment includes: hot start taq DNA polymerase, 2×PCR buffer, primer pair, probe and compound enzyme preparation;

[0060] Among them, 2×PCR buffer contains: 200mM potassium acetate, 20mM MgCl 2 , 50% glycerin, 25% DMSO, 0.5% Tween-20 and 5% PEG-200; pH is 8.3.

[0061] The compound enzyme preparation includes: 20% helicase, 15% lysozyme, 10% papain, 5% laccase, 10mmol / L Tris-HCL, 10mmol / L EDTA-2Na, 0.6% Triton X-100 and 2mmol / L citric acid Sodium, pH 7.2.

[0062] Compound enzyme preparation configuration steps:

[0063] 1. Preparation of 30% Triton X-100 stock solution: Mix 28.2ml of triton X-100 with 72.8mL of 0.1mol / l PBS (ph7.3), put it in a water bath at 37℃~40℃ for 2~3 hours, and make it fully Dissolve and mix well.

[0064] 2. Preparation of 0.05M Tris-hydrochloric acid buffer solution: Mix 50mL of 0.1M Tris-hydrochloride (Tris) solution with 44.7mL of 0.1M hydrochloric acid, add distilled water to m...

experiment example 1

[0088] Experimental group:

[0089] Experimental group: adopt the kit of embodiment 1, carry out fluorescent PCR amplification detection to 6 parts of known fungal infection positive samples (sample 1-6) according to the method of use in embodiment 1;

[0090] Control group: Replace the PCR buffer (2×) of the control group with ordinary PCR buffer (10×) as a control, and the others are the same as the experimental group; the composition of ordinary PCR buffer (10×) is as follows: 50mmol / L KCl, 2.5mmol / LMgCl 2 , 30mmol / L (NH 4 ) 2 SO 4 , 20 mmol / L Tris-HCl, pH8.3.

[0091] see results Figure 1A-Figure 1D , Figure 1A Middle: Curves 1, 5 and 7 are the fungal positive fluorescence (FAM) amplification curve, Candida positive fluorescence amplification curve (CY3) and Aspergillus negative signal fluorescence (VIC) amplification curve of sample 1 in the experimental group, respectively;

[0092] Curves 2, 4 and 7 are respectively the fungal positive fluorescence (FAM) amplif...

experiment example 2

[0101] As shown in the combination 1-6 of the following table 2, the same buffer solution is used to configure different composite enzyme preparations (the preparation method is the same as in Example 1, and different enzyme solution volumes are used according to the ratio of Table 2). For the same known fungus to be detected Infection-positive samples were detected, and the steps of the detection method and other reagents used were the same as in Example 1. After the detection was completed, the results were shown in figure 2 ( figure 2 Kinds of curves 1-13 represent combination 1-13 respectively), by analyzing the results, we can know that the optimum range of compound enzyme preparation configuration is respectively: helicase (15-25%), lysozyme (10-20%), papain ( 5-15%), laccase (0-10%); wherein the best ratio is combination 3: helicase (20%), lysozyme (15%), papain (10%), laccase (5%) ).

[0102] In addition, in order to investigate the comparison of the use effect of ...

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Abstract

The invention discloses a reagent set, a kit and a detection method for detecting fungal infection, and relates to the field of biotechnology. The reagent set comprises one of the following reagents or a combination of two of the following reagents: a PCR buffer and a complex enzyme preparation. The PCR buffer contains the following components: potassium acetate, MgCl2, glycerol, DMSO, Tween-20, and PEG-200. The complex enzyme preparation contains the following components: snail enzyme, lysozyme, papain and laccase. The reagent set can directly detect samples such as blood without the steps such as nucleic acid extraction and purification, and has the advantages of high sensitivity, good specificity and short detection time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reagent group, kit and detection method for detecting fungal infection. Background technique [0002] Hematological malignancies refer to malignant tumors of the blood system, including various types of leukemia, multiple myeloma, malignant lymphoma, and myelodysplastic syndrome. Due to the immune dysfunction of the body, the extensive use of glucocorticoids, cytotoxic drugs, and immune agents during treatment, the use of broad-spectrum antibiotics, and the increase in invasive procedures in these patients, invasive fungal disease (IFD) The incidence rate is gradually increasing. A recent multi-center epidemiological study in China shows that the incidence of patients in intensive care units accounts for about 8% to 15%, the incidence of organ transplant recipients is 20% to 40%, and the incidence of patients with hematological tumors Up to about 31%. [0003] Invasive fungal di...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/16C12Q2527/125C12Q2537/143C12Q2531/113C12Q2563/107
Inventor 许嘉森吴诗扬刘志明
Owner SUREXAM BIO TECH
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