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FCM (flow cytometry) based method for quickly and quantitatively detecting total viruses in freshwater environment

A flow cytometry and quantitative detection technology, which is applied in the field of rapid quantitative detection of total virus in freshwater environment based on flow cytometry, can solve the problems of time-consuming and laborious, and achieve the advantages of short detection time, simple operation steps and broad application prospects. Effect

Active Publication Date: 2012-07-18
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the time-consuming and labor-intensive problems of most existing methods, and to establish a detection method for rapid and quantitative determination of total virus content in freshwater environments using flow cytometry

Method used

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  • FCM (flow cytometry) based method for quickly and quantitatively detecting total viruses in freshwater environment
  • FCM (flow cytometry) based method for quickly and quantitatively detecting total viruses in freshwater environment
  • FCM (flow cytometry) based method for quickly and quantitatively detecting total viruses in freshwater environment

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Detection of virus total amount in a certain fresh water sample in Tianjin

[0020] 1. Pass the collected fresh water sample through a 0.22 μm filter membrane, add an appropriate amount of glutaraldehyde to fix it, so that the final mass concentration of glutaraldehyde is 0.25%, and immediately put it in a 4°C refrigerator; after 15 minutes, use liquid nitrogen Freeze and store in a -80°C freezer.

[0021] 2. Take out the sample frozen in the previous step and thaw it at room temperature, take 1ml into a sterilized centrifuge tube, add 10μl of Na with a concentration of 0.5mol / l 2 EDTA solution (final concentration up to 5mmol / l), and then add 5 μl of SYBR Green I stock solution (dilute 100-fold SYBR Green I stock solution with dimethyl sulfoxide passed through a filter membrane with a pore size of 0.22 μm as the stock solution).

[0022] 3. After shaking and mixing, stain in the dark at 80°C for 10 minutes. After cooling to room temperature, dilute with Mill...

Embodiment 2

[0025] The detection of virus total amount in embodiment 2 blank water sample

[0026] Milli-Q water was used as the water sample to be tested, and the method steps in Example 1 were used for detection. Since Milli-Q water does not contain viruses, bacteria, etc., the test results are as follows: figure 2 As shown, only the background part can be seen, and no virus colonies appear. This sample can be used as a negative control sample.

Embodiment 3

[0027] The detection limit of embodiment 3 method

[0028] Dilute a certain river water sample with Milli-Q water, and the diluted river water in each sample accounts for 0.05%, 0.1%, 0.5%, 1.0%, 10%, 25%, 50%, 80%, and 100% respectively .

[0029] After the above samples were processed according to the method in Example 1, they were detected by flow cytometry. The test results showed that ( image 3 ), the method among the present invention can detect the minimum concentration of virus in fresh water to be 4.04 * 10 4 counts / ml, the correlation of the method is very good, the correlation coefficient R 2 >0.99 (n=9), the repeatability of the method is very good, and the average error of the detected samples (n=9) is less than 10%.

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Abstract

An FCM based method for quickly and quantitatively detecting total viruses in a freshwater environment belongs to the field of biotechnology and water environment monitoring, and comprises six main steps including film filtration, settlement, storage, coloration, dilution and detection. The specific operation processes are as follows: passing a sampled water sample through a 0.22 micrometer filter film; adding glutaraldehyde, settling glutaraldehyde for 15 minutes at the temperature of 4 DEG C, and then refrigerating glutaraldehyde by liquid nitrogen; storing glutaraldehyde in a refrigerator at the temperature of minus 80 DEG C; adding Na2EDTA and fluorescent dye SYBRGreen I before detection, and performing coloration for 10 minutes at the temperature of 80 DEG C; diluting the sample by Milli-Q water after the sample cools down to the room temperature; and using the flow cytometer to detect. The method disclosed by the invention overcomes the problem that the present detection method for freshwater total viruses is time consuming and labor consuming, realizes quick and quantitative detection of the freshwater viruses, and has a wide application prospect in fields such as freshwater virus detection and aquatic bioecology study.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and water environment monitoring, and relates to a method for rapidly and quantitatively detecting total viruses in freshwater environments based on flow cytometry. Background technique [0002] Studies in the past 10 years have shown that there are a large number of viruses in the water environment. These viruses either infect the cells of aquatic organisms or float in the water, and are called planktonic viruses. [0003] Planktonic viruses are the most abundant biological group in water bodies. Planktonic viruses include bacteriophages (cyanophages), algae viruses, various animal and plant viruses free in water, and archaeal viruses in extreme water environments. The development and utilization of strategic biological resources such as planktonic viruses and the screening of algicidal viruses (cyanophages) are one of the biotechnological approaches to control and even eliminate the outb...

Claims

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Application Information

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IPC IPC(8): G01N15/14
Inventor 王莹莹马克・巴特兰姆马丽丽余辉
Owner NANKAI UNIV
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