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RCA method for detecting human herpesvirus-7 (HHV-7)

A technology of human herpes virus and HHV-7, which is applied in the direction of biochemical equipment and methods, and the determination/testing of microorganisms, which can solve the problems of high requirements for reaction conditions, low specificity and sensitivity of serological detection, and achieve high sensitivity , good specificity and simple operation

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; the PCR method has high sensitivity and specificity, but requires high reaction conditions, and there are also certain false positive reactions

Method used

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  • RCA method for detecting human herpesvirus-7 (HHV-7)
  • RCA method for detecting human herpesvirus-7 (HHV-7)

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-7 (HHV-7) positive specimen is processed according to the steps on the Qiagen company's virus DNA extraction kit operating instructions, and the DNA (viral genome 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 work...

Embodiment 2

[0042] A kind of specific detection of the RCA method that detects human herpesvirus-7 (HHV-7), 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-7 (HHV-7). 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 the electrophoresis result by a gel imager.The mehtod is a novel method for detecting human persesvirus -7 (HHV-7), which combines the padlock probe with RCA technology, the target gene sequence is specifically recognized by a padlock probe, and the detection signal is amplified through RCA amplification.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-7 (HHV-7). Background technique [0002] Human herpesvirus 7 (HHV-7) is a new type of human herpesvirus isolated from normal human peripheral blood mononuclear cells in 1990 following HHV-6. It has affinity for CD4+ lymphocytes in vitro and can Proliferation in PNA-stimulated human cord blood lymphocytes. Taxonomically, it belongs to the genus rosepesvirus of the subfamily Herpesviridae. HHV-7 has not yet been determined to be directly related to a certain disease. It may cooperate with HHV-6 to cause roseola in children, or activate HHV-6 virus to cause related diseases. HHV-7 is a ubiquitous human herpes virus, most of the antibodies are positive in infants 3 years after birth, and the antibody positive rate of healthy adults is over 95%. The transmission mode of HHV-7 is still unclear, but the virus mainly exists latently in periphe...

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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