High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5

A herpes virus and kit technology, applied in the field of high-sensitivity fluorescent quantitative PCR detection kits for herpes virus 4 and 5, can solve the problems of inability to report nucleic acid quantitative results, loss of nucleic acid, false negative test results, etc., and achieve optimal PCR reactions System and amplification procedures, avoid inaccurate detection results, and improve the effect of detection sensitivity

Inactive Publication Date: 2017-05-17
SHANGHAI XINGYAO MED TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is worth noting that many of the above-mentioned technical solutions only perform qualitative nucleic acid detection of herpes virus, and cannot report nucleic acid quantitative results. In particular, fluorescent PCR detection in the scheme does not use internal references. , or the loss of nucleic acid due to experimental operation errors will lead to "false negative" test results, resulting in missed tests and misdiagnosis, delaying patient treatment and recovery

Method used

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  • High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5
  • High-sensitivity quantitative detection kit for herpes virus 4 and herpes virus 5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the design of primer probe of herpes virus type 4, 5 nucleic acid fluorescent PCR quantitative detection kit

[0033] According to the HHV4, HHV5, and GAPDH gene sequences queried in the NCBI GenBank database, using Vector NTI, Oligo and other primer design software, the optimally obtained primer probe sequences are shown in Table 1, and the HHV4 primers amplify the virus BamHI-W gene The above 115bp fragment, the HHV5 primer amplified is the 91bp fragment on the IE2 gene of the virus, and the internal reference primer amplifies the 144bp fragment on the human GAPDH gene.

[0034] Table 1 Design kit primer probe

[0035]

[0036] The above primers and probes were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Embodiment 2

[0037] Embodiment 2: Herpesvirus 4, 5 type nucleic acid fluorescent PCR quantitative detection kit preparation

[0038] The reaction buffer of the kit is self-prepared. According to the concentration and volume of each component in Table 2, the reaction buffer of the kit is prepared for 32 people. The prepared reaction buffer is divided into 20 μl for each reaction. After adding 10 μl of the template, the total reaction volume is 30 μl.

[0039] Table 2 The volume of each component prepared by the reaction buffer of the kit

[0040]

[0041] The negative control of the kit uses normal human negative plasma; the quantitative calibrator is the cloning plasmid containing the amplified fragments of HHV4 and HHV5, which is diluted to 4 concentrations with TE buffer as the quantitative calibrator of the kit: 5.0x10 7 copies / ml, 5.0x10 6 copies / ml, 5.0x10 5 copies / ml, 5.0x10 4 copies / ml; the positive control is Escherichia coli containing the above cloned plasmid, diluted w...

Embodiment 3

[0045] Embodiment 3: the mensuration of kit detection sensitivity

[0046] (1) Herpes virus DNA extraction

[0047] Take herpes virus type 4 and 5 positive plasma with known concentration through clinical identification, adjust the concentration of the two viruses to the same level with normal human negative plasma and mix them in equal volumes. The concentration of herpes virus type 4 and type 5 in the mixed sample is 5x10 4 copies / ml). Then use normal human negative plasma to serially dilute the mixed sample 10 times to 5x10 3 copies / ml, 5x10 2 copies / ml, 5x10 1 copies / ml, draw 400 μl each of the above-mentioned virus dilution, the negative control of the kit, and the positive control, and use the magnetic bead method virus DNA extraction reagent in Example 2 to carry out virus DNA extraction on the nucleic acid extractor, and finally each sample obtained Nucleic acid each 50 μl.

[0048] (2) Fluorescent PCR detection

[0049] Take the reaction buffer of the kit in...

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Abstract

The invention relates to a kit for high-sensitivity quantitative detection of nucleic acids of a herpes virus 4 and a herpes virus 5 (HHV4 and HHV5) by using a fluorescent PCR technology. Specific nucleic acid sequences of the HHV4 and the HHV5 are amplified by using two pairs of primers separately, and an HHV4 DNA and an HHV5 DNA are quantitatively detected at different wavelengths through corresponding fluorescent probes; and meanwhile, an internal reference DNA is detected by using an endogenous gene specific primer and the fluorescent probes. Existence of the HHV4 DNA, the HHV5 DNA and the internal reference DNA is detected at the same time through a single-tube three-wavelength fluorescent PCR technology; the HHV4 DNA and the HHV5 DNA in a whole-blood sample and a plasma sample can be quantitatively detected; whether template loss caused by a PCR inhibitor or detection misoperation exists in each sample or not is judged through the detection result of an internal reference nucleic acid; and false negative leaking detection is reduced. The kit is simple and fast in operation, the quantitative result is sensitive and accurate, and the kit can be widely applied to quantitative detection of clinical infection of the herpes virus 4 and the herpes virus 5.

Description

technical field [0001] The invention relates to a high-sensitivity fluorescent quantitative PCR detection kit for herpesvirus types 4 and 5, belonging to the field of biotechnology. Background technique [0002] Herpesviruses are a group of medium-sized, enveloped, double-stranded DNA viruses. There are more than 120 known species. According to their physical and chemical properties, they are divided into four subfamilies: α, β, γ, and unclassified herpesviruses. They can infect humans and other vertebrates, mainly invading tissues derived from ectoderm, including skin, mucous membranes and nerve tissues. The sites of infection and the diseases they cause are various, and they have a tendency to latent infection. The common herpes viruses known to infect humans include human herpes virus types 1, 2, 3, 4, 5, 6, 7, and 8 (HHV 1-8). The disease can also be latent in human tissues for a long time. When the body's immune function is low, especially for leukemia and immune trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/10
CPCC12N15/1013C12Q1/686C12Q1/705C12Q2561/113C12Q2563/107C12Q2545/114C12Q2545/101
Inventor 吴大治夏懿吴梅
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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