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RCA method for detecting human parvovirus B19

A parvovirus, B19 technology, applied in the biological field, can solve the problems of inability to replicate, probes cannot be connected into a circle, etc., and achieve the effect of high sensitivity, good specificity, and simple operation

Inactive Publication Date: 2018-04-20
湖北朗德医疗科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Conversely, if there is a variation in the target sequence or there is no target gene to be detected, the probe cannot be connected into a circle, and replication cannot proceed

Method used

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  • RCA method for detecting human parvovirus B19
  • RCA method for detecting human parvovirus B19

Examples

Experimental program
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Effect test

Embodiment 1

[0027] An RCA method for detecting human parvovirus B19 is completed according to the following steps:

[0028] (1) Process the sample to be tested according to the steps of Qiagen's virus DNA extraction kit to obtain the DNA of the sample;

[0029] (2) Hybridization of capture probe: Add 6μl DNA sample, 1μmol / L lock probe 1μl, capture probe 1μl in 32μl hybridization buffer, mix well, hybridize in 55℃ water bath for 1h, slowly cool to room temperature, Add 40μ streptavidin-coated magnetic beads (dissolved in 2× binding buffer), mix well, place at room temperature for 20 minutes, and magnetically separate. Wash the magnetic beads twice with 1× binding buffer and discard the washing solution.

[0030] (3) Circular connection and restriction digestion of the padlock probe: 16μl deionized water, hybridization product prepared in step (2) and 2μl 10×E.coli Buffer, mix well, 95℃5min, 45℃incubate 40min Add 2μl of 10×BSA buffer and 0.2μl of E.coli DNA ligase, mix well, and carry out circul...

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Abstract

The invention discloses an RCA method for detecting human parvovirus B19. The method is implemented by the following steps: (1) extracting specimen DNA; (2) implementing RCA, then conducting gel electrophoresis by virtue of 1.0% agarose, and observing an electrophoresis result by virtue of a gel imaging instrument. The RCA method provided by the invention, which combines a padlock probe and an RCAtechnology, is a novel method for detecting the human parvovirus B19; by virtue of the padlock probe, the specific recognition of a target gene sequence is implemented; and by virtue of the RCA, detection signals are amplified. The method is free from the use of special equipment and the method is simple to operate, good in specificity and high in sensitivity; and the method has unique advantagesin the 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 parvovirus B19. Background technique [0002] Human parvovirus B19 (human parvovirus B19, HPV B19) belongs to the parvovirus family erythrovirus. It is a common rash disease in pediatrics—the cause of infectious erythema (also known as the fifth disease); B19 infects adults and can cause joints Inflammation or joint pain; B19 infection is mostly self-limiting for people with normal immunity; but for pregnant women and people with low immunity, B19 is more harmful. Infected pregnant women can cause fetal edema and fetal congenital anemia, and even stillbirth ; For those with weakened immunity, it can cause serious diseases such as chronic anemia and transient aplastic crisis. B19 may also be related to a variety of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Human parvovirus B19 infection is also one of the importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/682C12R1/93
CPCC12Q1/682C12Q1/6844C12Q2531/125C12Q2521/501
Inventor 朱世新朱玉华杨晓艳
Owner 湖北朗德医疗科技有限公司
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