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Detection kit for activity of cryptococcus and detection method

A technology of viability detection and detection method, applied in the field of medical biological detection, can solve the problems of bacterial viability judgment, unfavorable curative effect, low-level positivity, etc., and achieves the effect of simple use method, conducive to standardization and standardization

Inactive Publication Date: 2015-06-03
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the count of cryptococci in cerebrospinal fluid cannot judge the viability of the bacteria, and it is impossible to distinguish viable and non-viable cryptococci by light microscopy
However, the fungal culture method will prolong the culture time as the treatment time prolongs, which is not conducive to tracking the curative effect in real time.
Although the latex agglutination test can semi-quantitatively monitor the amount of cryptococcal bacteria, it has the possibility of hysteresis and false negatives. Some patients in clinical practice can still have a low-level positive blood latex agglutination titer after 1 year of regular antifungal treatment

Method used

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  • Detection kit for activity of cryptococcus and detection method
  • Detection kit for activity of cryptococcus and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: preparation kit of the present invention

[0046] Each component information of kit of the present invention is as follows

[0047] Detection solution A is 3.5% (w / v) CFW (Calcofluor white, purchased from Sigma-Aldrich, product number F3543) Preservation solution: 1.75mg CFW dissolved in 0.5ml double distilled water, NaOH to adjust the pH to 10, store in a light-proof tube .

[0048] The detection solution B is 1% (w / v) PI (Propidium Iodide, propidium iodide, Shanghai Future Reagents, product number A007233). Preservation solution: 0.5mg PI dissolved in 0.5ml double distilled water, and stored away from light.

[0049] The diluent C is: 2.6 g of disodium hydrogen phosphate, 0.43 g of sodium dihydrogen phosphate, and 8.18 g of sodium chloride, set the volume to 1000 ml, adjust the pH to 7.2, and take 50 ml for subpackaging.

Embodiment 2

[0050] Example 2: Detection of cryptococcal activity in cerebrospinal fluid

[0051] 1. Preparation of related reagents

[0052] Related reagents are prepared with reference to Example 1

[0053] 2. Detection process

[0054] 2.1 During the routine inspection of cryptococcal meningitis cerebrospinal fluid, take 5ml of cerebrospinal fluid sample, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 200 μl of diluent C, resuspend, and make the sample to be tested;

[0055] 2.2 Shake 200 μl of the resuspended sample and perform flow cytometry zero adjustment ( figure 1 A);

[0056] 2.3 Add 5 μl of detection solution A to the sample after zero adjustment in 2.2, and test again on the machine, the sample volume is 30 μl, avoid cell debris, select a normal-sized cell population for analysis, and use FL1 and FL4 channels to detect fluorescence;

[0057] 2.4 Add 5 μl of detection solution B to the sample in 2.3, test again on the machine, record at least 10,000 cells...

Embodiment 3

[0061] Embodiment 3: Sensitivity and specificity detection of detection method of the present invention

[0062] 1. Preparation of related reagents

[0063] Related reagents are prepared with reference to Example 1

[0064] 2. Experimental sample preparation

[0065] 2.1 Take 0.5ml of peripheral blood from the mouse by the orbital blood collection method, lyse the red blood cells with the erythrocyte lysate, resuspend them in diluent C after centrifugation at 1000rpm, and adjust the white blood cells to 1×10 with a hemocytometer 6 / ml.

[0066] 2.2 After resuspending the cultured cryptococcus with diluent C, dilute to 1×10 6 / ml bacterial suspension. Take 0.5ml of the bacterial suspension at this concentration for later use, and then take another 0.5ml of the bacterial suspension to inactivate in a 90°C water bath for 10 minutes. Mix the distribution of normal bacteria suspension and inactivated bacteria suspension with the white blood cell suspension in 2.1 at a ratio of...

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Abstract

The invention relates to the technical field of medical biological detection and provides a detection kit for the activity of cryptococcus. The kit adopts two types of fluorescent dyes to mark mycothallus and the activity of fungus respectively, and can analyze the quantity and the activity of the cryptococcus in cerebrospinal fluid fast and accurately by utilizing the kit and by means of assistance of a common flow cytometry. The invention also provides a detection method utilizing the detection kit for the activity of the cryptococcus and has important guiding significance for conducting prognosis to a patient and making the clinical treatment scheme.

Description

technical field [0001] The invention relates to the technical field of medical biological detection, and relates to a kit for detecting the vitality of cryptococcus and a detection method thereof. Background technique [0002] Cryptococcus neoformans is an important pathogenic fungus that can cause cryptococcal meningitis and disseminated cryptococcal infection. The treatment time of cryptococcal meningitis is long, and the recurrence rate is high. One of the most important problems plaguing clinicians in the treatment of cryptococcal meningitis is the lack of an objective time standard for judging the course of treatment due to the inability to make an effective judgment on the vitality of cryptococcal bacteria during treatment (Hong Wei, Wen Hai, Chen Bin , Liao Wanqing. Comparative study on mycological indicators of cryptococcal meningitis cerebrospinal fluid [J]. Chinese Journal of Dermatology, 2003,08:48.). [0003] According to the Infectious Diseases Society of Amer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
Inventor 潘搏廖万清潘炜华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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