Human herpesvirus type 6 real-time fluorescent quantitative PCR typing detection kit

A technology of real-time fluorescence quantification and detection kits, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve problems such as difficult typing, long time required, and low sensitivity

Inactive Publication Date: 2015-10-07
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional herpes virus detection methods mainly include virus isolation and culture and serological detection. The former was once the "gold standard" for the diagnosis of herpes virus, but it takes a long time (3-30 days) and has low sensitivity (especially after antiviral drug treatment). ), typing difficulties and other defects; the latter is divided into direct antigen detection method and antibody indirect detection method. The direct antigen detection method has low sensitivity because some viruses do not produce detectable antigens, and the indirect antibody detection method is due to herpes virus It takes about 1 week after infection to produce an effective concentration of antibodies, making early diagnosis impossible, and there are cross-reactions between different herpes viruses, and the specificity of antibodies is not high

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  • Human herpesvirus type 6 real-time fluorescent quantitative PCR typing detection kit
  • Human herpesvirus type 6 real-time fluorescent quantitative PCR typing detection kit

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Experimental program
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Effect test

Embodiment 1

[0027] see figure 1 , the human herpesvirus type 6 real-time fluorescent quantitative PCR typing detection kit provided by the present invention consists of quantitative PCR reaction solution 1, HHV-6A standard product 2, HHV-6B standard product 3, HHV-6A positive control product 4, HHV -6B consists of 5 positive control substances, 6 negative control substances, 7 instructions and 8 boxes.

[0028] The quantitative PCR reaction solution contains PCR buffer, MgCl 2 , dNTPs, thermostable DNA polymerase, upstream amplification primer, downstream amplification primer, HHV-6A fluorescent probe and HHV-6B fluorescent probe.

[0029] The primer sequences for PCR amplification are:

[0030]Upstream amplification primer (SEQ ID No: 1): 5'-CCGTGAAGTTGGGGGATGAG-3';

[0031] Downstream amplification primer (SEQ ID No: 2): 5'-CAGAAGCAGCAATCGCAACAC-3';

[0032] Fluorescent probes and fluorescent markers for the detection of each herpesvirus are:

[0033] HHV-6A fluorescent probe (SEQ ...

Embodiment 2

[0039] Example 2 Sensitivity and specificity test of human herpesvirus type 6 real-time fluorescent quantitative PCR typing detection kit

[0040] (1) Materials:

[0041] The selected pathogenic microorganisms include: experimental group: HHV-6B (GS strain) provided by the Microbiology Teaching and Research Group of Nanjing Medical University, HHV-6A screened from clinical blood samples by Children's Hospital Affiliated to Zhejiang University; control group: Staphylococcus aureus, Escherichia coli Bacteria, Hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome were provided by the Bacteria Room, Virus Room and Gene Amplification Room of Children's Hospital Affiliated to Zhejiang University.

[0042] (2) Design and synthesis of primers and probes:

[0043] Conduct bioinformatics analysis on the conserved region sequences of HHV-6A and HHV-6B, design and screen PCR amplification primers and specific fluorescent probes, and entrust Shanghai Sangon Bio...

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Abstract

The invention provides a real-time fluorescent quantitation PCR (polymerase chain reaction) parting detection kit for human herpesvirus-6 (HHV-6). The real-time fluorescent quantitation PCR parting detection kit consists of quantitation PCR reaction liquid, an HHV-6A standard substance, an HHV-6B standard substance, an HHV-6A positive reference substance, an HHV-6B positive reference substance, a negative reference substance, a specification and a kit body, wherein the quantitation PCR reaction liquid contains a PCR buffer solution, MgCl2, dNTPs, heat-resistant DNA polymerase, an upstream amplification primer, a downstream amplification primer, an HHV-6A fluorescent probe and an HHV-6B fluorescent probe. By the adoption of a real-time fluorescent quantitation PCR technology and a double-color fluorescent probe, the kit disclosed by the invention can detect two subtypes of the HHV-6 by a one-step method; the HHV-6A and HHV-6B in the sample can be simultaneously parted; a positive virus subtype can be accurately quantified in real time; the urgent need of early and acute parting diagnosis on the HHV-6 infection can be met; a basis is supplied to epidemiological investigation and targeted treatment on the HHV-6 infection.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a fluorescent quantitative PCR detection kit, in particular to a dual-probe real-time fluorescent quantitative PCR detection and typing of human herpesvirus 6 (human herpesvirus 6, HHV-6) diagnostic kit, which can simultaneously type and quantify the two subtypes of HHV-6, HHV-6A and HHV-6B. Background technique [0002] Herpesviridae is a group of medium-sized, enveloped DNA viruses, more than 100 species have been discovered. Herpesviruses known to be associated with human disease include herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Marr virus (EBV), human giant Cytovirus (HCMV), human herpesvirus types 6, 7 and 8 (HHV-6, HHV-7, HHV-8). Among them, HHV-6 belongs to the β-herpesvirus subfamily and can be divided into two subtypes, 6A and 6B. It is a relatively newly discovered human herpesvirus and is the pathogen of acute eruption...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 尚世强杜立中舒强陶然李伟
Owner ZHEJIANG UNIV
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