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RCA method for detecting human herpesvirus-6 (HHV-6)

A technology of human herpes virus and HHV-6, which is applied in the direction of biochemical equipment and methods, microbiological determination/testing, etc., can solve the problems of low specificity and sensitivity of serological detection, high requirements for reaction conditions, and achieve specificity Good, high sensitivity, simple operation effect

Inactive Publication Date: 2019-05-31
湖北朗德医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The specificity and sensitivity of serological detection are not high, often resulting in false positive results; cell isolation and culture of virus and PCR methods have high sensitivity and strong specificity, but the reaction conditions are high, and there are also some false positive reactions

Method used

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  • RCA method for detecting human herpesvirus-6 (HHV-6)
  • RCA method for detecting human herpesvirus-6 (HHV-6)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The detection of the minimum detection concentration of the RCA amplification reaction is completed according to the following steps:

[0027] (1) The known human herpesvirus-6 (HHV-6) positive specimen is processed according to the steps on the Qiagen company's viral DNA extraction kit operating instructions, and the DNA (viral genomic DNA) of the sample is obtained, and the ultra-micro spectrophotometer is used Measure the DNA concentration (NanoDrop 2000C), and dilute it to detect templates with final concentrations of 5pM, 50pM, 500pM and 5nM, respectively;

[0028](2) Hybridization of capture probes: Add 6 μl of DNA samples diluted in different concentrations in step (1), 1 μl of 1 μmol / L padlock probe, and 1 μl of 1 μmol / L capture probe in the hybridization reaction tube, and mix well , hybridized in a water bath at 55°C for 1 h, cooled slowly to room temperature, and added 40 μl of streptavidin-coated magnetic beads dissolved in 2× binding buffer (2×: 2 times wor...

Embodiment 2

[0042] A kind of specific detection of the RCA method that detects human herpesvirus-6 (HHV-6), completes according to the following steps:

[0043] (1) Process the sample to be tested according to the steps in the instruction manual of Qiagen's viral DNA extraction kit to obtain the DNA (viral genomic DNA) of the sample;

[0044] (2) Hybridization of capture probes: add 6 μl of DNA sample, 1 μl of 1 μmol / L padlock probe, and 1 μl of 1 μmol / L capture probe to the hybridization reaction tube, mix thoroughly, hybridize in a water bath at 55°C for 1 hour, and cool slowly to At room temperature, add 40 μl of streptavidin-coated magnetic beads dissolved in 2× binding buffer (2×: 2 times working concentration, magnetic beads were purchased from Shanghai Bioengineering Co., Ltd.), mix well, place at room temperature for 20 min, and magnetically separate. Wash the magnetic beads twice with 1× binding buffer (1×: 1 times working concentration), and discard the washing solution.

[00...

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Abstract

The invention discloses an RCA method for detecting human herpesvirus-6 (HHV-6). The RCA method is completed according to the following steps: (1) DNA extraction of a specimen; (2) hybridization of acapture probe; (3) cyclization connection and enzyme digestion of a padlock probe; and (4) RCA amplification, agarose gel electrophoresis and observation of electrophoresis results by a gel imager. The method is a novel method for detecting human herpesvirus-6 (HHV-6), which combines the padlock probe withtheRCA technology, the target gene sequence is specifically recognized by the padlock probe,and the detection signal is amplified through RCAamplification.The method does not need special equipment, has simple operation, has good specificity and high sensitivity, and has unique advantages inthe aspect of virus detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RCA method for detecting human herpesvirus-6 (HHV-6). Background technique [0002] Human herpesvirus type 6, belonging to the β subfamily of the Herpesviridae family, is a new virus that was cultured from lymphocytes of patients with lymphoproliferative diseases and AIDS in 1986. The virus has the morphological characteristics of typical herpes viruses, HHV-6 is similar to the human cytomegalovirus (HCMV / HHV-5) host, and mainly infects CD4+T cells, CD8+T cells, monocyte-macrophages and NK Cells can also be latent in salivary glands, mammary glands, and kidneys and continue to replicate at low densities. When infecting monocyte-macrophages, the host cells will not appear cytopathic, but the viral DNA can remain in the cells for a long time, forming a recessive infection. HHV-6 can be transmitted through saliva droplets and close contact (such as kissing, sharing meals and drinki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
Inventor 朱世新朱玉华石康
Owner 湖北朗德医疗科技有限公司
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