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General-purpose nucleic acid isothermal detection reagent for adenovirus associated with respiratory tract infection
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A detection reagent, adenovirus technology, applied in the biological field, can solve the problem of lack of RPA detection reagents, etc., and achieve the effect of rapid screening
Active Publication Date: 2019-12-13
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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For common adenovirus infections, there are currently no RPA assays available
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Embodiment 1
[0036] The design of embodiment 1, RPA primer and probe
[0037] Download the hexon gene sequences of adenovirus types 3, 7, 11, 14, and 55 from the Genbankdatabase of NCBI, compare and analyze all gene sequences with MEGA5.0, and select conserved regions for primer and probe identification design. A total of 2 sets of primer probes were designed and synthesized, and the sequences are shown in Table 1 below.
[0038] Table 1 is the primer probe sequence
[0039]
Embodiment 2
[0040] Embodiment 2, establishment and optimization of RPA reaction system and method
[0043] Adenovirus types 3, 7, 11, 14, and 55 (Specialized Committee of Infectious Diseases of the Army, Guidelines for Diagnosis and Treatment of Adenovirus Infection, Medical Journal of the PLA, 2013(8), 529-534; Huang Guohong, New Types of Adenovirus Research progress, Acta Virology, 2013 (29), 342-348) were inoculated with Hep 2 cells (purchased from ATCC, CAT.CCL 10), and placed at 37 degrees CO 2 Cultured in an incubator, and then observed the cytopathic changes every day. When the proportion of cytopathic cells in the culture flask exceeds 75%, the culture flask is frozen and thawed once at -70°C, and then the cell culture supernatant containing the virus is aliquoted.
[0044] Detect the titer of each adenovirus: The culture supernatants of adenovirus types 3, 7, 11, 14, and 55 were diluted serially 10 time...
Embodiment 3
[0080] Example 3, the application of RPA primers and probes in the detection of samples to be tested
[0082] Throat swab specimens were collected from 10 cases of respiratory-related virus infection, 5 cases of adenovirus type 7 (numbered S1, S2, S3, S4 respectively; 3 cases of adenovirus type 55 (numbered respectively S5, S6, S7), influenza virus Infected 3 cases (numbered respectively S8, S9, S10), the samples S1-S7 nucleic acid were confirmed to be positive by adenovirus real-time fluorescent quantitative PCR detection reagent, and the nucleic acid of S8-S10 sample was negative. DNA / RNA extraction kit was used to extract Viral nucleic acid in the sample.
[0083] 2. RPA amplification
[0084] The viral nucleic acid obtained from each sample in the above-mentioned one was used as an amplification template, and the primer probe set 2 was used for RPA amplification. The reaction system and conditions were as described in the second-1 of t...
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Abstract
The invention discloses a general nucleic acid isothermal detection reagent of respiratory tract infection associated adenoviruses. The invention provides recombinasepolymerasenucleic acid detection set nucleic acid which is used for identifying respiratory tract infection adenoviruses and is composed of a primer pair and a probe; the primer pair is composed of a single-stranded DNA (Deoxyribonucleic Acid) molecule represented by a sequence 1 in a sequence table and a single-stranded DNA (Deoxyribonucleic Acid) molecule represented by a sequence 2 in the sequence table; the probe is composed of A-dt-FAM-tetrahydrofuran-dt-BHQ1-B, wherein the nucleotide sequence of A is a sequence 3; the nucleotide sequence of B is a sequence 4. An experiment testifies that general RPA (RecombinasePolymeraseAssay) detection primer and probe are designed aiming at six types of the respiratory tract infection associated adenoviruses; an RPA detection system and method for the respiratory tract infection associated adenoviruses are established; the technology can be used for carrying out field rapid screening and detection on the respiratory tract infection associated adenoviruses, has an important value and can realize rapid screening of unknown samples.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a general-purpose nucleic acid isothermal detection reagent for adenovirus associated with respiratory tract infection. Background technique [0002] Adenovirus is a non-enveloped DNAvirus belonging to the genus Mammalian Adenovirus of the family Adenoviridae. Virus particles are spherical and icosahedral in three-dimensional symmetry. Divided into 6 subgroups (A-F), including more than 50 genotypes. [0003] Adenovirus can infect the respiratory tract, gastrointestinal tract, urinary tract, bladder, eye, liver, etc. About 1 / 3 of the known serotypes of human adenovirus are usually associated with human diseases, but one serotype can cause different clinical diseases ; On the contrary, different serotypes can also cause the same disease. [0004] In clustered respiratory infections, adenovirus is one of the main pathogens. The main types include 3 types, 7 types, 11, 14, 55 and o...
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