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Primer, probe, reagent kit and method for detecting human parvovirus B19 based on real-time fluorescence PCR technique

A human parvovirus and technical detection technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of insufficient coverage, low accuracy, easy missed detection of B19 virus, etc., to achieve The effect of reducing labor and material costs and reducing the amount of samples used

Pending Publication Date: 2020-04-03
SUZHOU YAOMING KANGDE INSPECTION TESTING
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] None of the reported B19 virus qPCR detection methods can guarantee coverage of all types of B19, and the coverage is insufficient. When used for B19 virus detection, it is easy to miss detection, the accuracy is low, and the result reliability is low.

Method used

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  • Primer, probe, reagent kit and method for detecting human parvovirus B19 based on real-time fluorescence PCR technique
  • Primer, probe, reagent kit and method for detecting human parvovirus B19 based on real-time fluorescence PCR technique

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Embodiment Construction

[0070] The technical solution of the present invention will be clearly and completely described below, obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0071] 1. Take 1x10 7 HEK293 cells were collected by centrifugation at 5000rpm for 5 minutes.

[0072] 2. Resuspend the cells with 0.5 mL PBS. Cell DNA was extracted with Qiagen DNeasy Blood and Tissue Kit.

[0073] 3. Use Nanodrop to detect DNA OD260 and OD280 absorption values, calculate the cellular DNA concentration, and adjust the concentration to 0.1 μg / μL

[0074] 4. Serially dilute DNA template 1 and template 2 to contain 1x10 per 5 μl 5 , 1x10 4 , 1x10 3 , 1x10 2 and 10 copies of the target sequence

[0075] 5. Prepare the reac...

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Abstract

The invention provides a primer, probe, reagent kit and method for detecting human parvovirus B19 based on a real-time fluorescence PCR technique. The primer and the probe consist of two groups: a first group including a template 1 as shown in SEQID NO:7, a forward primer 1 as shown in SEQID NO:1, a backward primer 1 as shown in SEQID NO:2 and a probe 1 as shown in SEQID NO:5, and a second group including a template 2 as shown in SEQID NO:8, a forward primer 2 as shown in SEQID NO:3, a backward primer 2 as shown in SEQID NO:4 and a probe 2 as shown in SEQID NO:6. In accordance with the human parvovirus B19 of all different types, corresponding forward and backward primers, corresponding probes and a reaction procedure are designed, a real-time quantitative PCR method having the advantagesof being good in specifity, good in sensitivity, high in spreadability of variants and the like is established, and the detection method is suitable for heavy and complicated detection of mass production of biological products.

Description

technical field [0001] The invention belongs to the field of biological detection, which uses an in vitro nucleic acid detection method to detect virus contamination in biological products, and in particular relates to a primer, probe, kit and method for detecting human parvovirus B19 by using real-time fluorescent PCR technology. Background technique [0002] Human parvovirus B19 (Human Parvovirus B19) is a non-lipid-enveloped single-stranded DNA virus that can cause various diseases in humans, and belongs to the family Parvoviridae and the genus Rhodovirus. The virus is mainly transmitted through respiratory secretions, blood and blood products. The population infection rate is as high as 60%-70%. When pregnant women are infected with the virus, it is easy to cause hydrops fetalis and congenital anemia, which can lead to fetal death in severe cases; when children are infected with the virus, chronic hemolytic anemia or infectious erythema will appear. In recent years, a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107
Inventor 董元舒刘存昌苏财忠童涌
Owner SUZHOU YAOMING KANGDE INSPECTION TESTING
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