GB/T 14926.25-2026 Laboratory animal—Method for examination of reovirus 3（Reo3） English, Anglais, Englisch, Inglés, えいご
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ICS 13.220.10
CCS H 57
National Standard of the People's Republic of China
GB/T 14926.25-2026
Replaces GB/T 14926.25-2001
Laboratory animal - Method for examination of reovirus 3（Reo3）
实验动物 呼肠孤病毒Ⅲ型检测方法
Issue date: 2026-01-28 Implementation date: 2027-02-01
Issued by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China
the Standardization Administration of the People's Republic of China
Contents
Foreword
Introduction
1 Scope
2 Normative References
3 Terms and Definitions
4 Principle
5 Reagents and Equipment
6 Detection Methods
7 Result Interpretation
8 Result Reporting
Laboratory Animals — Detection Method for Reovirus Type 3
1 Scope
This document describes detection methods for reovirus type 3 (Reo3).
This document applies to the detection of Reo3 antibodies and nucleic acid in mice, rats, hamsters, guinea pigs, their related products, and environmental samples from laboratory animal facilities.
2 Normative References
The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 14926.50 Laboratory animal – Enzyme-linked immunosorbent assay
GB/T 14926.51 Laboratory animal – Immunoenzyme assay
GB/T 14926.52 Laboratory animal – Immunofluorescence assay
3 Terms and Definitions
No terms and definitions are required for this document.
4 Principle
4.1 Principle of Antibody Detection
Based on immunological principles, Reo3 antigen is used to detect Reo3 antibodies in the serum of mice, rats, hamsters, and guinea pigs.
4.2 Principle of Nucleic Acid Detection
Specific primers and probes are designed and synthesized based on the conserved gene sequence of the Reo3 virus S3 protein to amplify target fragments. Real-time quantitative reverse transcription polymerase chain reaction (real-time quantitative RT-PCR, RT-qPCR) is used to detect whether Reo3 nucleic acid components are present in the sample.
5 Reagents and Equipment
5.1 Reagents
5.1.1 Enzyme-linked Immunosorbent Assay (ELISA) Antigen
5.1.1.1 Specific Antigen
Infect BSC-1 or BHK21 cells with Reo3 virus. When cytopathic effect (CPE) reaches +++ to ++++, harvest the culture. Freeze-thaw three times or sonicate. Remove cell debris by low-speed centrifugation. Concentrate the supernatant by ultracentrifugation to prepare the ELISA antigen.
5.1.1.2 Normal Antigen
Supernatant obtained by freeze-thawing and disrupting uninfected BSC-1 or BHK21 cells, followed by low-speed centrifugation to remove cell debris.
5.1.2 Antigen Slides
Infect BHK21 cells with Reo3 virus and culture for 4 to 5 days. When CPE reaches ++ to +++, disperse the cells with trypsin, wash with phosphate-buffered saline (PBS), and prepare smears. Air dry at room temperature while simultaneously irradiating with UV light at a distance of 20 cm for 30 minutes. Fix with cold acetone for 10 minutes. Store at -20°C.
5.1.3 Positive Serum
Antiserum obtained by immunizing specific pathogen free (SPF) mice, rats, guinea pigs, and hamsters with Reo3 virus antigen.
5.1.4 Negative Serum
Serum from SPF mice, rats, guinea pigs, and hamsters.
5.1.5 Enzyme Conjugate
Horseradish peroxidase-labeled goat or rabbit anti-mouse, rat, guinea pig, hamster IgG antibodies, used for detecting corresponding animal serum antibodies. Horseradish peroxidase-labeled Staphylococcal Protein A (SPA) can be used for detecting serum antibodies in mice, guinea pigs, and hamsters.
5.1.6 Fluorescent Conjugate
Fluorescein isothiocyanate-labeled goat or rabbit anti-mouse, rat, guinea pig, hamster IgG antibodies, used for detecting corresponding animal serum antibodies.
5.1.7 Nucleic Acid Detection Reagents
5.1.7.1 DEPC-treated RNase-free water or commercially available RNase-free water.
5.1.7.2 Commercially available RNA extraction kit.
5.1.7.3 Real-time quantitative RT-PCR master mix: commercially available reagent.
5.1.7.4 Positive control: Inactivated tissue sample or cell culture containing Reo3 virus nucleic acid.
5.1.7.5 Negative control: Tissue sample or cell culture not containing Reo3 virus nucleic acid.
5.1.7.6 Primers and probes: Synthesize primers and probes according to the sequences in Table 1. Prepare primers and probes as 10 μmol/L stock solutions. Store at -20°C or below.
5.2 Equipment
5.2.1 Microplate reader.
5.2.2 Fluorescence microscope.
5.2.3 Ordinary light microscope.
5.2.4 37°C incubator or water bath.
5.2.5 Real-time quantitative PCR instrument.
5.2.6 Centrifuge.
5.2.7 Biosafety cabinet.
5.2.8 Clean bench (laminar flow hood).
5.2.9 Other equipment: Micropipettes, centrifuge tubes, polystyrene microplates, PCR tubes/plates, etc.
6 Detection Methods
6.1 Use the ELISA method for serological detection, operating according to GB/T 14926.50.
6.2 Use the indirect immunofluorescence assay (IFA) method for serological detection, operating according to GB/T 14926.52.
6.3 Use the immunoenzyme assay (IEA) method for serological detection, operating according to GB/T 14926.51.
6.4 Use the real-time quantitative RT-PCR method for Reo3 virus nucleic acid detection, operating according to Appendix A.
