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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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 e

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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Example Embodiment

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

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

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

[0022] 3. After shaking and mixing, dye for 10 minutes at 80℃ and avoid light. After cooling to room temperature, dilute with Milli-Q water until the concentration detecte...

Example Embodiment

[0025] Example 2 Detection of Total Virus in Blank Water Sample

[0026] The Milli-Q water was used as the water sample to be tested, and the test was performed according to the method steps in Example 1. 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 community appears. This sample can be used as a negative control sample.

Example Embodiment

[0027] The detection limit of the method in Example 3

[0028] Dilute a river water sample with Milli-Q water. After dilution, the amount of river water in each sample accounted for 0.05%, 0.1%, 0.5%, 1.0%, 10%, 25%, 50%, 80%, 100%. .

[0029] After the above samples were processed according to the method in Example 1, they were tested on a flow cytometer. The test results show that ( image 3 ), the method of the present invention can detect the lowest concentration of virus in fresh water is 4.04×10 4 counts / ml, the correlation of the method is very good, the correlation coefficient is R 2 >0.99 (n=9), the method has good repeatability, and the average error of the test sample (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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