Fluorescence quantitative PCR technology for extracting and amplifying hepatitis B virus nucleic acid by 'one tube method'

Abstract

The invention discloses a trace nucleic acid releasing agent. The releasing agent realizes that hepatitis B virus nucleic acid extraction and fluorescent augmentation are directly performed in the same tube, radically solves the problems of nucleic acid loss and pollution caused by a multi-step nucleic acid extraction method, and is applicable to the fluorescent PCR detection of a whole blood sample for the first time; the releasing agent adopts quantificational provirus serum as a quantificational standard and realizes the whole process monitoring of the standard to the sample; the releasing agent adopts double pairs of forward primers and reverse primers and a probe decorated by forward fluorescence and reverse fluorescence to perform fluorescent PCR augmentation, further to improve the sensitivity and the augmentation efficiency of the releasing agent; blue promoting fluorescer is introduced to ensure that prepared PCR operating liquid is light blue, and has the functions of steady fluorescent signals, strengthened augmentation efficiency and so on, without affecting the fluorescent detection; the releasing agent sets two temperature gradient programs, adopts a rear-section fluorescence acquisition method, is convenient to analyze and improves the service life of an instrument; compared with the prior method, the technology has revolutionary innovation.

Description

Technical field [0001] The patent of the invention belongs to nucleic acid extraction and fluorescent polymerase chain reaction (PCR) quantitative technology; this technology realizes that the two steps of nucleic acid extraction and subsequent PCR amplification are carried out in the same PCR tube, which fundamentally solves the pollution of this type of experiment problem and achieve truly fast and accurate quantification. Background technique [0002] At present, the HBV DNA fluorescence quantitative PCR technology that is being clinically applied in the domestic and foreign markets mainly has the following characteristics: [0003] 1. Nucleic acid extraction: There are mainly the following types: 1) Polysaccharides are concentrated to precipitate pathogens, then add alkaline lysate to pyrolyze and centrifuge to obtain nucleic acid; 2) Alkaline lysate is directly mixed with pathogen-containing liquid, and then pyrolyzed , centrifugation to obtain nucleic acid; 3) Chromat...

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Problems solved by technology

[0004] The above extraction methods must go through 3 to 8 steps of changing tubes, centrifugation (negative pressure suction), pipetting and other steps to obtain nucleic acids. All of the above processes have quantitative errors or errors caused by factors such as nucleic acid loss, contamination, and fatigue operations.

[0005] 2. Quantitative standard products outside: Most of the standard products are pure DNA products (recombined or cloned products) provided by the reagent company together with the kit. They do not participate in nucleic acid extraction at the same time as the specimen, but directly enter PCR amplification. Some kits provide Nucleic acid is extracted from the standard and the sample at the same time, but the nucleic acid incorporation method is used instead of the original virus particles, which is fundamentally different from the sample, and it is impossible to truly and fully monitor the nucleic acid extraction and amplification process of the sample

[0006] 3. Primers and probes: a single forward primer, a fluorescently labeled probe and a single reverse primer are used to form a specific amplification sequence, and there is still room for improvement in its amplification efficiency and sensitivity

[0007] 4. PCR working reaction solution: PCR amplification buffer, primers, probes, Taq DNA polymerase and other required components are mixed into a PCR working solution, which is a colorless liquid, which is easy to cause operational errors caused by visual fatigue

[0008] 5. Setting of PCR amplification temperature conditions: The traditional 40 amplification cycles are used to collect fluorescence signals during the annealing period, but there is no positive signal in the first 10 amplification cycles of PCR, and there is a signal null test; In addition, the fluorescent signal is unstable in the first few cycles of fluorescent PCR, which is not conducive to the analysis of results.