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Real-time quantitative PCR detection kit for herpes simplex virus type 1

A technology of herpes simplex virus and detection kit, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., and can solve the problems of low diagnostic value of clinical etiology, high false positive/false negative, and positive Low detection rate and other issues, to achieve good clinical application value, strong specificity, and high sensitivity

Pending Publication Date: 2020-12-11
海南省百维恩生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although virus culture and isolation is considered to be the "gold standard" for detecting virus infection, it is time-consuming and has low sensitivity. It requires specialized laboratories for virus culture and identification, which is costly, expensive, and has a low positive detection rate, etc.
Electron microscopy can observe the physical morphology of viruses in infected specimens. The process is cumbersome and expensive, and requires specialized institutions, equipment, and technicians to implement, and it has low sensitivity, is prone to sampling errors, and has high false negatives. It is only suitable for Observation of scientific research, not suitable for diagnosis of clinical etiology
Immunological antigen detection method has low specificity, high false positive / false negative, and relatively low value for clinical etiological diagnosis
Because serological testing is indirect speculation, the actual reference value is very low

Method used

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  • Real-time quantitative PCR detection kit for herpes simplex virus type 1
  • Real-time quantitative PCR detection kit for herpes simplex virus type 1
  • Real-time quantitative PCR detection kit for herpes simplex virus type 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0042] Implementation Case 1. Preferred HSV1-specific Primers and Probes

[0043] The design of primers and probes is based on the NCBI genome database, using MegAlign, Primer and other software to select HSV1 conserved and specific sequences. Since the HSV1 DNA sequence has high homology with other herpesviruses (HSV2, CMV, EBV, VZV, HHV6, HHV7, HHV8) that infects humans, and human genome DNA is unavoidable when extracting HSV1 DNA, when designing primers, the primers There are at least 6 base mismatches with other herpes-like viruses and human genome sequences, and at least 3 base mismatches at the 3' end.

[0044] The probe adopts TAGMAN probe, the 5' end is marked with a fluorescent reporter group of 6FAM, and the 3' end is marked with a fluorescent quencher group of BHQ1. The annealing temperature of the probe is about 10°C higher than the annealing temperature of the primer to ensure that the probe preferentially binds to the template. The probe is close to the upstrea...

Embodiment example 2

[0047] Implementation case 2. Standard preparation of recombinant plasmid of HSV1 target gene

[0048] 1) According to the selected primers, the target gene sequence of HSV1 was downloaded from NCBI, and the PCR amplification sequence was synthesized by the company (Thermo Fisher), and the recombinant plasmid (pUC57-T vector) of the synthetic product was constructed to transform E. coli DH5α.

[0049] 2) Plasmid amplification and extraction:

[0050] (1) Inoculate 10 μL of Escherichia coli in solid LB medium containing ampicillin, culture upside down, and overnight at 37°C;

[0051] (2) Pick a single colony and inoculate it in 5ml liquid LB medium, and culture it on a horizontal shaker at 37°C, 200rpm, for 12-16h;

[0052] (3) Take the cultured bacterial solution by shaking, centrifuge at 5000g for 5 minutes, discard the supernatant; add 2ml Buffer P1, shake or pump, and mix well until no cell clumps gather;

[0053] (4) Add 2ml Buffer P2, mix by inverting slightly, incubate...

Embodiment example 3

[0069] Implementation case 3. HSV1 qRT-PCR detection kit

[0070] The components of the qRT-PCR kit are shown in Table 3.

[0071] Table 3. HSV1 qRT-PCR detection kit components

[0072]

[0073]*The positive control of the UL40 recombinant plasmid with 5 concentration gradients of known copy number can be used to make a standard curve using the method of Example 2, and the viral load of the nucleic acid sample can be calculated.

[0074] The reaction system of the kit is shown in Table 4.

[0075] Table 4. qRT-PCR reaction system

[0076] 2×PCR Mix 10μL upstream primer 0.2 μL downstream primer 0.2 μL probe 0.2 μL Tear fluid nucleic acid sample / positive control / negative control 2μL RNase-free water 7.4μL total 20 μL

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Abstract

The present invention discloses a real-time quantitative PCR detection kit for herpes simplex virus type 1. The detection kit takes a UL40 gene of HSV1 as a target and comprises specific primers and aspecific probe of the HSV1, a positive control, a negative control, 2xPCR Mix and RNase-free water. By collecting tear from eyes of patients and scraping tissues, aqueous humor orvitreous humor and other specimens from corneal infection parts, thereal-time quantitative PCR detection kit can rapidly and accurately form an etiological detection, at the same time can monitor virus loads in real time, thus realizes accurate diagnosis and treatment of the patients, and has characteristics of high sensitivity, high specificity, economy, simplicity, convenience and rapidness.

Description

technical field [0001] The invention belongs to the technical field of virus detection and relates to a PCR detection kit, in particular to a real-time quantitative PCR detection kit for infectious eye disease herpes simplex virus type 1. Background technique [0002] Herpes simplex virus type 1 (HSV1) is a common viral pathogen. According to the World Health Organization (WHO), in 2015, more than 3.7 billion people under the age of 50 (ie 67% of the population) were infected with HSV1[1]. HSV1 is the most common pathogen in infectious eye diseases, and can infect almost all ocular tissues (including eyelids, conjunctiva, cornea, uvea, and retina) to cause diseases [2]. Clinically, the incidence of HSV1 infectious eye disease is much higher than that of other pathogens such as bacterial, fungal or other less common pathogenic eye diseases. Globally, the morbidity and blindness rate of infectious eye diseases are very high. For HSV1 keratitis alone, the incidence rate reache...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/705C12Q2561/101C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 姚玉峰许叶圣郑利斌
Owner 海南省百维恩生物科技有限责任公司
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