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Real-time isothermal amplification-based kit for human papilloma virus E6/E7 gene detection, and application thereof

A technology for human papillomavirus and gene detection, which is applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as the difficulty in designing viral nucleic acid molecules, and achieve improved detection sensitivity and high amplification efficiency , strong specific effect

Active Publication Date: 2017-06-13
西安迪安医学检验实验室有限公司
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AI Technical Summary

Benefits of technology

This patented detective assay uses an enzyme called DNA polymerase that helps create copies of genetic material from patient samples more efficiently than traditional methods like gel electrophoresis or Western blotting. It also includes various techniques such as fluorimetric analysis (FCA), flow cytometry, histochemistry, immunofluoresence, chemiluminescent microscopy, etc., which are used together to make accurate diagnoses based on how well they work at different stages during treatment. Overall, this technology allows research scientists to accurately determine if there's any disease associated with these treatments without having them wait longer periods before being able to find out what kind of therapy works best against it.

Problems solved by technology

The technical problem addressed in this patented patents relating to the current state of knowledge regarding how to effectively diagnose various diseases associated with human papilloma epithelial cells called Papilomaviridae, particularly those causing cervicitis syndrome due to certain etiological agents like Human Immunodeficiency Virus Type-1 strains. Current assays such as qPCR cannot provide sufficient accuracy while avoiding false negative responses during multiple tests. There is a desire to establish a quick and reliable way to specifically recognize these disease indicators and determine their presence in order to improve clinically important decisions made earlier.

Method used

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  • Real-time isothermal amplification-based kit for human papilloma virus E6/E7 gene detection, and application thereof
  • Real-time isothermal amplification-based kit for human papilloma virus E6/E7 gene detection, and application thereof
  • Real-time isothermal amplification-based kit for human papilloma virus E6/E7 gene detection, and application thereof

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Embodiment 1

[0062] Embodiment 1: The composition and main components of the kit for real-time NASBA detection of high-risk HPV E6 / E7 mRNA of the present invention are as follows:

[0063]

Embodiment 2

[0064] Example 2: Design of real-time NASBA primers and probes

[0065] Firstly, the whole genome DNA sequences of 14 high-risk HPV types were downloaded from the gene bank of NCBI in the United States, and the E6 / E7 gene sequences were found at the same time, and the primers and probes were designed in combination with the software Oligo 7.56 (Molecular Biology Insights, Inc. USA). The design idea is: comprehensively consider the specificity and generality of the detection of 14 high-risk HPV E6 / E7 genes (degenerate bases are used at the viral nucleic acid mutation sites), and the general principles that should be followed in the design of primers and probes ( Such as Tm value, 3' end free energy, GC content, avoidance of internal structure and dimer formation, etc.), and the influence of the reverse primer plus the promoter sequence that can be recognized by T7 RNA polymerase, etc. After the designed primers and probes are synthesized, the constructed plasmids are used as te...

Embodiment 3

[0073] Embodiment 3: the preparation of positive quality control product and negative quality control product

[0074] 1. Preparation of positive quality control

[0075] A commercially available commercial nucleic acid extraction kit was used to extract viral RNA from samples infected with monotype HPV virus and perform PCR amplification. The amplified target band was recovered, ligated with the modified pET32-MS3his vector, ligated overnight at 4°C, the ligated product was transformed into BL21(DE3)pLysS competent cells, resistance screening, shaking, PCR identification For positive cases, the recombinant plasmid was extracted and verified by sequencing. The cloned bacterial liquid containing the target fragment was induced and expressed by the IPTG inducer, and the induced bacterial liquid was collected and ultrasonically crushed to separate the supernatant and precipitate. The collected supernatant was a pseudovirus solution, that is, a positive quality control product. ...

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Abstract

The invention discloses a real-time isothermal amplification-based kit for human papilloma virus E6/E7 gene detection, and application thereof. A primer and a probe are designed by aiming at 14 high-risk HPV, and rapid detection and identification on HPV E6/E7 mRNA can be realized specifically. The kit provided by the invention has the characteristics of high speed, high efficiency, sensitivity, specificity, real-time detection and analysis and the like, provides a rapid and accurate molecular detection method for generation investigation of HPV and prevention and treatment of cervical cancer, greatly reduces the inspection cost and has an important popularization and application value.

Description

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Claims

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Application Information

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Owner 西安迪安医学检验实验室有限公司
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