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Virus titration method

A virus and virus count technology, applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., can solve problems such as hindering PCR, long separation time, expensive instruments, etc.

Inactive Publication Date: 2011-03-02
JAPAN TOBACCO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] On the other hand, as a method of increasing the concentration of viruses in a solution and concentrating viruses, for example, ultracentrifugation can be cited, but this method requires expensive equipment and a long separation time, and requires a lot of labor in the operation.
In addition, there are methods of concentrating the virus by precipitating the virus with ammonium sulfate, polyethylene glycol, etc., but because the reagents used in these methods will hinder the subsequent PCR for virus detection, there is a problem that it must be carried out after virus precipitation. Sample purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] Example 1 Exploration of lectins capable of detecting HHV

[0226] Biotinylated lectin was reacted with a solution of cultured HHV-6, and it was examined whether HHV-6 can be concentrated by binding to magnetic beads bound with tamavidin.

[0227] 1. Making HHV-6 Solution from Cultured T Cells

[0228] Human umbilical cord blood-derived cultured T cells were infected with recombinant HHV-6 (patent No. 3923505) expressing EGFP to prepare an EGFP-type HHV-6 solution.

[0229] 2. Preparation of tamavidin magnetic beads

[0230]Wash 300 μl of magnetic beads (Dynabeads M-270 Carboxylic Acid, Dynal Company) coated with carboxyl groups on the surface with 300 μl of 0.01N sodium hydroxide for 10 minutes, and then wash 3 times with 300 μl of ultrapure water, each time for 10 minutes. For the washed magnetic beads, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) dissolved in cold ultrapure water to make the final concentration to 0.2M, shake at room...

Embodiment 2

[0239] The quantification of HHV-6 in the saliva of embodiment 2 not using reference virus

[0240] HHV-6 concentrations in saliva were quantified using biotinylated lectin and tamavidin magnetic beads without reference virus.

[0241] 1. Collection of saliva

[0242] Saliva from the test subjects was collected using a saliva collection tube (Salivette Cotton, manufactured by Sarstedt). Immediately before saliva collection, the subjects rinsed their mouths with distilled water twice, and then held the cotton core of the saliva collection tube in the oral cavity for 2 minutes to collect saliva.

[0243] 2. Quantification of HHV-6 by Traditional Methods

[0244] First, HHV-6 concentrations in saliva were quantified using conventional methods.

[0245] For the 400 μl of saliva collected in the above-mentioned project 1, use BioRobot EZ1 (QIAGEN Company) and EZ1 Virus Mini Kit v2.0 (QIAGEN Company), according to the protocol of EZ1 Virus MiniHandbook (QIAGEN Company), HHV...

Embodiment 3

[0257] The quantification of HHV-6 in the saliva that embodiment 3 uses reference virus to carry out

[0258] For HHV-6 concentrations in saliva, quantification was performed using biotinylated lectin, tamavidin magnetic beads, and reference virus.

[0259] 1. Saliva collection method

[0260] Saliva from the test subjects was collected using a saliva collection tube (Salivette Cotton, manufactured by Sarstedt). Immediately before saliva collection, the subjects rinsed their mouths with distilled water twice, and then held the cotton core of the saliva collection tube in the oral cavity for 2 minutes to collect saliva.

[0261] 2. Quantification of HHV-6 using traditional methods

[0262] Quantification of HHV-6 in saliva was carried out by the same method as in Item 2 of Example 2. Moreover, the virus experiments in this experiment were carried out by extremely skilled experimenters. The results are shown in Table 1.

[0263] 3. Determination of HHV-6 using bioti...

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Abstract

Provided is a method for counting human herpes virus (HHV) harvested from body fluids and a kit for performing said method. In the past, skilled technique was required to accurately count HHV from body fluids. Provided is a novel titration method that allows simpler, and more accurate and efficient measurement of HHV counts in body fluids. Since the method can tolerate continuous evaluation of HHV counts in body fluids, it can also be used for quantitative evaluation of accumulated fatigue.

Description

technical field [0001] The present invention relates to a method for quantifying the number of human herpesviruses (HHV) collected from body fluids, and a kit for performing the method. Background technique [0002] The accumulation of "fatigue" can cause many serious problems such as death from overwork, suicide, and lifestyle-related diseases. However, compared with other fields, the scientific or medical research on "fatigue" is obviously lagging behind, and no breakthrough has been found to solve the problems that "fatigue" brings to personal health and social life. [0003] The lack of objective measurement of "fatigue" can be cited as a major cause of the delay in the research on "fatigue" and the development of preventive and therapeutic methods. In particular, with regard to "accumulation of fatigue" and "long-term persistent fatigue" that have the greatest impact on life and health, the measurement methods have not yet been established, and there is an urgent need ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06C12Q1/68G01N33/569
CPCG01N2333/36G01N2333/03G01N33/56983
Inventor 高仓由光市川雅子近藤一博清水昭宏
Owner JAPAN TOBACCO INC
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