By vantal Evaluation and application of real-time fluorescence quantitative RT-PCR in dengue mouse modelAs can be seen from the new data, suture kit The market influence is also growing, and the product share is also relatively increasing, which has great potential in the future. https://pinnaclemedics.com/
At present, an ideal animal model of severe dengue fever has not been established in China, and there is a lack of highly instructive, reliable and stable pathogenic indicators. Literature reports point out that the quantitative detection of virus can be aimed at virus particles, virus antigens, virus nucleic acids and so on. Traditional methods include virus isolation, cell culture isolation and neonatal rat intracranial inoculation isolation, etc. This method is reliable, but the operation is complicated and the experimental period is long, which is not conducive to the rapid diagnosis of dengue virus. Followed by antigen detection methods, enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (GICA) and immunofluorescence (IFA). However, the antigen detection method is complicated to operate, and the detection sensitivity is low, which is easy to cross-react with other flavivirus and produce false positive. In the past few decades, molecular diagnostic technology has been continuously improved, which makes the detection and identification of various pathogens more reliable. Among them, real-time fluorescence quantitative PCR has gradually become a new gold standard for rapid diagnosis of viruses. In this study, the existing RT-PCR detection methods of dengue virus were optimized, and a one-step real-time fluorescence quantitative RT-PCR method was established, which was used to evaluate the mouse model of dengue virus infection.
Rodent surgical instruments
1 materials and methods
1.1 experimental materials
1.1.1 Experimental Animals 20 SPF BALB/c male suckling mice, weighing 2-3g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. [SCXK (Beijing) 2019-0008″ target=_blank> for the amplification of dengue virus. Rodent surgical instruments.
Type I interferon receptor knockout mice (Ifnar1-1-)[SCXK (Beijing) 2014-0011″ target=_blank> with clean C57BL/6 background for 4-6 weeks, with 40 males and females, weighing 18-22g, were provided by Northern Center of Medical Laboratory Animal Institute of China Academy of Medical Sciences for the preparation of dengue mouse model. The animal experiment scheme was approved by the Committee on the Use and Management of Laboratory Animals of China Academy of Medical Sciences (WW18002). Animal feeding and experiments were conducted in the Biosafety Secondary Animal Laboratory (ABSL-2) of the Institute of Medical Experimental Animals, China Academy of Medical Sciences, following the 3R principle. Rodent surgical instruments.
1.1.2 virus strain
Dengue virus (DENV): Type I (TH-Sman strain), Type II (NewGuinea strain /NGC adaptive strain (TH-36 strain), Type II (TH87 strain) and Type IV (H241 strain) were all purchased from ATCC and preserved by our research group. The virus strains used for specificity analysis include: enterovirus 71 EV71(MP10 strain), Coxsackievirus 10 CA10(SJZK12-0046T strain), Coxsackievirus 16 CA16(Shzh05- 10,000 strains of mouse pneumonia virus PVM(15 strains), murine hepatitis virus MHV(A59 strain) and reo (reo). Rodent surgical instruments.
1.2 Main reagents and instruments
RNA extraction kit (RNeasyMiniKit:52904) and real-time fluorescence quantitative PCR kit (ProbeRT-PCRKit:204443); AppliedBiosystems7500 real-time fluorescence quantitative PCR instrument.
1.3 experimental methods
1.3.1 Primers and probes
Rodent surgical instruments
According to literature [4″ target=_blank>, specific primers and TaqMan probes were selected, and the upstream and downstream primer and probe sequences were: 5′-Garagaccagatcctgctgtct-3′; 5¨-ACCATTCCATTTTCTGGCGTT-3¨; 5′-FAM-AGCATCATTCCAGGCAC-MGB-3’。 Rodent surgical instruments.
1.3.2 preparation of standard.
Select the target fragment of 3’UTR of dengue virus type II (NewGuinea strain) to prepare the standard. PCR amplification was carried out using the above specific primers, the target gene was cloned into TOPO vector, and the plasmid was constructed as a standard. The above operations were entrusted to Sino-American Taihe Company. After the plasmid was synthesized, the original standard was determined by ultraviolet spectrophotometer, and the plasmid concentration was 1035 ng/μ L.