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Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments

A pseudovirion and nucleic acid fragment technology, which is applied in the field of preparation of artificial binary pseudovirions containing HCV and HIV nucleic acid fragments, can solve problems such as difficult degradation, excessive deviation of quantitative results, and raising the entry threshold of molecular diagnostic reagents. , to achieve the effect of high clone purity, simple method and good stability

Inactive Publication Date: 2014-05-14
东北制药集团辽宁生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for easier production of high-purity noncontamined copies (pseudo viral) from different sources like blood or urine samples that contain both human immunodeficiency virus type 1(Human Immunodirma Virus Type I), Human T Cell Lymphotropic Herpesviruses types 31A/B, Different strains of Hepatitis B Virus). These pseudo vires are stable even when mixed together but still have excellent properties making them useful tools for medical purposes.

Problems solved by technology

Technically speaking, it's difficult to prepare an artificially made pseudo vircular particle containing DNA segments from different sources due to instability caused during storage or transportation processes. Current methods involve complicated steps such as extracting samples with solvents like ethanol followed by purification procedures before analysis. Additionally, current techniques require expensive equipment and specialized expertise, making their applications limited.

Method used

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  • Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments
  • Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments
  • Preparation method of artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments

Examples

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Effect test

Embodiment 1

[0028] Testing of Standard Products of HCV Fluorescence Quantitative PCR Kit

[0029](1) Preparation of HCV working standard

[0030] The pseudovirus particles prepared by the above method were first quantified using the purchased commercial HCV fluorescent quantitative PCR detection kit, and then according to the quantification results, the pseudovirus was diluted in multiples with negative serum to prepare a working standard 1-4. The specific concentration is 1x10 7 IU / ml, 1x10 6 IU / ml, 1x10 5 IU / ml and 1x10 4 IU / ml.

[0031] (2) Extraction of HCV working standards and samples

[0032] Use our company's nano-magnetic bead method nucleic acid extraction reagent to extract the working standard, the extraction process is as follows:

[0033] a. Take an appropriate amount of 1.5ml centrifuge tubes, mark the working standard 1-4 and the sample to be tested respectively, add 400μl lysate to each tube, the lyse solution is composed of sodium lauryl sulfate 0.5-2.5%, Triton X-...

Embodiment 2

[0051] Testing of HIV Fluorescent Quantitative PCR Kit Standards

[0052] (1) Preparation of HIV working standard

[0053] The pseudovirus particles prepared by the above method are first quantified using the purchased commercial HIV fluorescent quantitative PCR detection kit, and then according to the quantification result, the pseudovirus is diluted with negative serum to prepare a working standard 1-4. The specific concentration is 1x10 7 IU / ml, 1x10 6 IU / ml, 1x10 5 IU / ml and 1x10 4 IU / ml.

[0054] (2) Extraction of HIV working standards and samples

[0055] Use our company's nano-magnetic bead method nucleic acid extraction reagent to extract the working standard, the extraction process is as follows:

[0056] a. Take an appropriate amount of 1.5ml centrifuge tubes, mark the working standard 1-4 and the sample to be tested respectively, add 400μl lysate to each tube, the lyse solution is composed of sodium lauryl sulfate 0.5-2.5%, Triton X-1000.5 -2.0ml / 100ml, guani...

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Abstract

The invention relates to a preparation method of an artificial dual false virus particle comprising HCV (hepatitis C virus) and HIV (human immunodeficiency virus) nucleic acid fragments, belonging to the field of the clinical diagnosis of the biomedicine. The preparation method comprises the steps of designing and synthesizing the forward and reverse primers of coliphage MS2 mature protein gene, envelope protein gene and Pac site, taking MS2 bacteriophage RNA as a template, carrying out RT-PCR (reverse transcription-polymerase chain reaction) amplification by the primers, and finally connecting an amplified product onto a pMD18-T carrier for sequencing to test and verify the clone correctness; extracting the PMD 18-T plasmid comprising the MS2 mature protein gene, the envelope protein gene and the Pac site, separating the inserted mature protein gene, envelope protein gene and Pac site by Ncol and BamHI restriction enzymes, and connecting to a Ncol and BamHI bi-digested pET-28b carrier to obtain a connected product. The preparation method of the artificial dual false virus particle comprising HCV and HIV nucleic acid fragments is simple and easy to operate, and the obtained false virus is high in clone purity, and is an ideal raw material of a job standard product and a quality control product of an HCV and HIV diagnostic kit.

Description

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Claims

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Application Information

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Owner 东北制药集团辽宁生物医药有限公司
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