Kit and extraction method for rapidly and conveniently extracting viral DNA from body fluid

Abstract

The invention discloses a kit and extraction method for rapidly and conveniently extracting viral DNA from body fluid. The kit comprises four components, wherein a first component comprises 2mol / L-6mol / L of guanidinium isothiocyanate, 0.5%-12% of TritonX-100, 10mmol / L-300mmol / L of tris(hydroxymethyl)aminomethane and 5%-15% of PEG4000; a second component contains 1mmol / L to 50mmol / L of sodium dihydrogen citrate, 0.005mmol / L to 0.05mmol / L of tris(hydroxymethyl)aminomethane, 5% to 30% of PEG4000 and 0.005% to 0.05% of magnetic beads; a third component contains 10mmol / L to 200mmol / L of sodium dihydrogen citrate and 1% to 10% of TritonX-100; and a fourth component comprises mineral oil. The method for extracting DNA by using the kit can enter a PCR link through splitting decomposition / combination and cleaning, and compared with a conventional magnetic bead extraction method, required manual operations and waiting time are less; a reagent applied to extraction is efficient and safe, and thus, the harm to a human body is avoided to the greatest extent; and the sensitivity is further improved as the DNA in a sample can be completely applied to PCR amplification.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a kit and an extraction method for quickly and conveniently extracting viral DNA in body fluids. Background technique [0002] Nucleic acid molecular biology techniques can be used in commonly used methods such as infectious disease source detection, genetic disease detection, transgenic detection, phylogenetic evolution, and origin of species. Various current nucleic acid molecular biology techniques, including fluorescent quantitative PCR, digital PCR, and next-generation sequencing (NGS), have made a lot of progress. However, the good detection results of these techniques are all based on the premise of the efficiency of nucleic acid purification and recovery. Good nucleic acid purification technology should be simple, safe and cheap, and the recovered nucleic acid should reach sufficient concentration and purity. [0003] In general, nucleic acid is...

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Problems solved by technology

[0006] Nucleic acid purification by magnetic beads is often accompanied by uninterrupted mixing, shaking, etc., which is easy to implement with instruments, but it is difficult to meet the requirements if manual operation is required, which leads to a serious drop in recovery efficiency, which is a common problem in most magnetic bead recovery kits at present.

And usually, the DNA recovered by the magnetic beads needs to be eluted at a certain temperature (such as 70°C) by the eluent. In order to improve the elution efficiency or compensate for the loss caused by evaporation, a larger eluent volume is often required. , leading to a relatively low final nucleic acid concentration, such as when the sample DNA content itself is low, it may reduce the final detection sensitivity

[0007] In general, magnetic beads are strong inhibitors of PCR. Directly adding the PCR solution in the state of binding DNA will seriously inhibit the fluorescence intensity of the fluorophore in real-time fluorescent quantitative PCR, resulting in false negative results.

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