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Human parvovirus and humanbocavirus double real-time fluorescent PCR detection primer, probe and reagent kit, and detection method

A technology of human bocavirus and human parvovirus, applied in the biological field, can solve problems such as false negatives, achieve high specificity, prevent false negative and false positive results, and have high sensitivity

Inactive Publication Date: 2019-12-31
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Real-time fluorescence quantitative polymerase chain reaction technology (RT-PCR) is realized by real-time monitoring of cumulative fluorescence intensity through the quantitative starting point and fluorescence detection system. It has the advantages of high sensitivity, strong specificity, good linear relationship, and simple operation. It has been widely used It is used in rapid detection, quantitative analysis, early diagnosis, genotyping, etc. of human and animal diseases, but it is prone to false negatives in the application

Method used

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  • Human parvovirus and humanbocavirus double real-time fluorescent PCR detection primer, probe and reagent kit, and detection method
  • Human parvovirus and humanbocavirus double real-time fluorescent PCR detection primer, probe and reagent kit, and detection method
  • Human parvovirus and humanbocavirus double real-time fluorescent PCR detection primer, probe and reagent kit, and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Select the specific detection gene or conserved sequence of human parvovirus B19V and human bocavirus HBoV: select the conserved gene VP1 for B19V, and select the conserved gene NS1 for HBoV. Find the full-length sequences of genes and conserved regions in Genbank, import several full-length sequences of genes or sequences into the sequence comparison software, and compare and analyze to obtain conserved sequence segments that can be used for primer probe design. The conserved sequence segments of each gene were input into the primer design software, and three primer probes for detection targets were designed. During the design process, the problem of co-amplification of primers and probes of different target genes in one reaction system should be fully considered. Therefore, the consistency of Tm value and the uniformity of GC content should be considered in the design of primers and probes, and at the same time, hairpins should be avoided as much as possible. Structur...

Embodiment 2

[0062] 1. Genome extraction

[0063] Whole blood samples were collected, and after centrifugation, serum or plasma was collected for nucleic acid extraction using a commercial kit.

[0064] 2. Preparation of reaction system

[0065] The reaction system detected by the kit is 20 μL, and its configuration is as follows: 10×PCR buffer 2 μL; Hot Start DNA Polymerase (5U / μL) 0.2 μL; UDG (2U / μL) 0.1 μL; dN(U)TP (25mM) 0.16 μL ; MgCl 2 (25mM) 2.4μL; IAC template 1μL; 10×primer-probe mixture 2μL, wherein, the concentration of the primer is 2-8μM, the concentration of the probe is 1-3μM; DNA template 5μL, ultrapure water to 20μL, of which The probe is the primer probe obtained in Example 1.

[0066] 3. PCR reaction

[0067] Put the PCR tube into the fluorescence quantitative PCR instrument, and carry out the PCR reaction according to the following procedure: 50°C, 2min; 95°C, 5min; 95°C, 15s, 55°C, 45s, cycle 45 reactions.

[0068] 4. The specific judgment methods of the results a...

Embodiment 3

[0073] Minimum detection limit test

[0074] Select the samples of B19V and HBoV, and gradiently dilute the templates to be equivalent to 10 7 PFU / mL, 10 6 PFU / mL, 10 5 PFU / mL, 10 4 PFU / mL, 10 3 PFU / mL, 10 2 PFU / mL, 10PFU / mL test sample. Using the kit of the present invention to detect templates with different dilutions of B19V, the minimum detection limit test results for the kit to detect the target virus are shown in Table 3:

[0075] Table 3 The minimum detection limit test results of the kit for detecting B19V

[0076]

[0077] As can be seen from Table 3, the minimum detection limit of the kits for the target virus reached 10 3 PFU / mL.

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Abstract

The invention belongs to the field of application of biologic techniques, and particularly relates to a human parvovirus and humanbocavirus double real-time fluorescent PCR detection primer, probe andreagent kit, and a detection method. The effects of quantitative detection, high sensitivity, high specificity and false negative prevention are achieved, a complete solution is provided for quick quantitative detection of human parvovirus B19V and humanbocavirus in clinical samples, and quick accurate quantitative detection of the human parvovirus B19V and the humanbocavirus can be realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double real-time fluorescent PCR detection primer, probe, kit and detection method for human parvovirus and human bocavirus. Background technique [0002] At present, human parvovirus B19V and human boca virus HBoV infection mainly exist in children in my country, both of which can be transmitted through the respiratory tract and are one of the parvoviruses. Parvoviruses are non-enveloped icosahedral viruses approximately 22 nm in diameter that contain a linear single-stranded DNA genome. Parvoviridae, one of the smallest DNA viruses known to infect mammalian cells, is divided into two subfamilies, Parvovirinae and Densovirinae, which infect vertebrates and invertebrate cells. At present, according to the number of open reading frames, transcription localization, autonomous or helper virus-assisted replication, sequence homology and other factors of vertebrate virus p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2545/101C12Q2563/107
Inventor 杨海英岳素文马寅佳王雷李英张志强
Owner 北京卓诚惠生生物科技股份有限公司
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