Adenovirus multi-fluorescent quantitative PCR (polymerase chain reaction) detection kit and using method thereof

Abstract

The invention discloses an adenovirus multi-fluorescent quantitative PCR (polymerase chain reaction) detection kit, comprising PCR liquid, a primer probe combination, a positive quality control sample, and a negative quality control sample. The adenovirus multi-fluorescent quantitative PCR detection kit also comprises liquid for nucleic acid extraction and an inner reference substance for nucleic acid extraction, wherein the inner reference substance is inactivated T4 bacteriophage; the primer probe combination comprises a primer probe (general: SEQ ID NO4-6; subtype SEQ ID NO7-9) for adenovirus nucleic acid and a primer probe (SEQ ID NO1-3) for the inner reference substance; the 5' terminals of the SEQ ID NO3, 6, 9 are connected to a fluorescent report group; and the 3' terminals are connected to a fluorescent quenching group. A using method of the kit is also disclosed by the invention. Viral nucleic acid extraction is carried out by the liquid for nucleic acid extraction before the fluorescent quantitative PCR detection; and the inner reference substance is added in the extraction process of the viral nucleic acid. The kit and the using method thereof disclosed by the invention have the characteristics of being convenient to use, and accurate in detection result; all adenoviruses can be detected; and B and E subgroups can be parted.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an adenovirus multiplex fluorescent quantitative PCR detection kit and a use method thereof. Background technique [0002] Adenovirus (adenovirus) is a particle with a diameter of 70-90nm without an envelope, which is composed of 252 capsomers arranged in an icosahedron. The capsid contains 240 hexads, 12 pentads and 12 cilia, in addition to other small proteins such as VI, VIII, IX, IIIa and IVa2. The hexaplex is the main protein that forms the 20 triangular faces of the virus capsid. The top of the 12 is a complex composed of 5 pentad subunits and 3 ciliary proteins. Protruding, the tips of the cilia form the scolex. The pentad and the scolex region of the cilium can bind to the virus receptor on the cell surface and play a very important role in the process of virus infection of cells. [0003] Since the discovery and successful isolation of adenovirus in the 1950s, more th...

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Problems solved by technology

[0008] Whether it is a competitive internal standard or a non-competitive internal standard, there are mainly three forms: 1) Plasmid or DNA, RNA fragments: This form of internal standard is easy to prepare, but it is often unstable, and the plasmid is easy to form aerosol, It is easy to cause pollution in the PCR process, especially because it is a DNA fragment, which often does not participate in the sample extraction process and is only added in the process of fluorescent quantitative PCR, so it can only monitor the PCR process, but not the quality of sample extraction.

The current papers on adenovirus fluorescent quantitative PCR detection reported in the literature mainly use plasmids as internal reference substances or quality controls; As an internal reference, it can monitor the release of virus particles and the efficiency of nucleic acid extraction, making up for the lack of plasmid DNA or naked DNA, RNA fragments as internal reference or quality control products, but due to the difficulty in the preparation and transportation of conventional virus particles, and for some Virus particles (HBV and HCV) of patient specimens also have ethical issues, so they are less used; 3) virus-like particles: certain viruses such as MS2 bacteriophage, filamentous phage, Lamda phage and tobacco mosaic virus (TMV) The shell can withstand the action of RNase and DNase, so exogenous target sequences can be wrapped into the shell of these viruses, which can protect them from the degradation of RNase and DNase in the environment

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