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RCA method for detecting herpes simplex virus-2(HSV-2)

A herpes simplex virus, HSV-2 technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low specificity and sensitivity of serological detection and high requirements for reaction conditions, and achieve specificity. Good, high sensitivity, simple operation effect

Pending Publication Date: 2019-06-07
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 herpes simplex virus-2(HSV-2)
  • RCA method for detecting herpes simplex virus-2(HSV-2)
  • RCA method for detecting herpes simplex virus-2(HSV-2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The detection of the minimum detection concentration of the RCA amplification reaction is completed according to the following steps:

[0028] (1) The known herpes simplex virus-2 (HSV-2) positive specimen is processed according to the steps on the Qiagen company's viral DNA extraction kit operating instructions, and the DNA (viral genome DNA) of the sample is obtained, and is analyzed with an ultra-micro spectrophotometer Measure the DNA concentration (NanoDrop 2000C), and dilute it to detect templates with final concentrations of 5pM, 50pM, 500pM and 5nM, respectively;

[0029](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...

Embodiment 2

[0046] A specific detection of the RCA method that detects herpes simplex virus-2 (HSV-2), completes according to the following steps:

[0047] (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;

[0048] (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.

[0049] (...

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Abstract

The invention discloses an RCA method for detecting herpes simplex virus-2(HSV-2). The method is completed according to the following steps of (1) extracting specimen DNA; (2) catching hybridization of a probe; (3) performing cyclized connection and enzyme cutting of a loctal probe; and (4) performing RCA amplification, then performing electrophoresis with agarose gel, and observing an electrophoresis result through a gel imager. A method combining the loctal probe with an RCA technique is a new method for detecting the herpes simplex virus-2(HSV-2), through the loctal probe, a target gene sequence is specifically recognized, and a detection signal is enlarged through RCA amplification. According to the method, special equipment is not needed, the operation is simple, the specificity is good, the sensitivity is high, and the method has unique advantages in the respect of virus detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RCA method for detecting herpes simplex virus-2 (HSV-2). Background technique [0002] Herpes simplex is a viral skin disease caused by herpes simplex virus, which is called heat sore in traditional Chinese medicine. Can cause a variety of human diseases, such as gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infection and neonatal infection. After infecting the host, latent infection is often established in nerve cells, and asymptomatic detoxification will occur after activation, maintaining the transmission chain in the crowd, and the cycle repeats itself. Herpes simplex belongs to the subfamily Aviridae of the family Herpesviridae, and the size of the virus plasmid is about 180 nanometers. According to the difference in antigenicity, the virus is currently divided into type 1 and type 2. The nucleotide sequences of the two types of viruses have 50...

Claims

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Application Information

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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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