GB/T 14926.19-2026 Laboratory animal—Method for examination of Hanta virus (HV) 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 14926.19-2026
Replaces GB/T 14926.19-2001
Laboratory animal - Method for examination of Hanta virus (HV)
实验动物 汉坦病毒检测方法
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 Main Reagents and Equipment
6 Detection Methods
7 Result Interpretation
8 Result Reporting
Laboratory Animals — Detection Method for Hantavirus
1 Scope
This document describes methods for detecting Hantavirus antibodies and nucleic acid.
This document applies to the detection of antibodies and nucleic acid in laboratory animals such as mice and rats.
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 14926.50-2001 Laboratory animal – Enzyme-linked immunosorbent assay
GB/T 14926.52-2001 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, Hantavirus antigen is used to detect Hantavirus antibodies in the serum of mice and rats.
4.2 Principle of Nucleic Acid Detection
Specific primers and probes are designed and synthesized based on conserved gene sequences of Hantavirus to amplify target fragments. Real-time quantitative reverse transcription polymerase chain reaction [real-time quantitative RT-PCR (RT-qPCR)] is used to detect whether Hantavirus nucleic acid components are present in the sample.
5 Main Reagents and Equipment
5.1 Reagents
5.1.1 Enzyme-Linked Immunosorbent Assay (ELISA) Antigen
5.1.1.1 Specific Antigen
Inside a biosafety cabinet, use E6 cells infected with Hantaan virus or Seoul virus. When specific fluorescence reaches +++, harvest the culture. Inactivate with β-propiolactone, 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 E6 cells, followed by low-speed centrifugation to remove cell debris.
5.1.2 Antigen Slides
Inside a biosafety cabinet, use E6 cells infected with Hantaan virus or Seoul virus. Replace maintenance medium every 2 to 3 days and culture for 7 to 10 days. Determine specific intracellular fluorescence using the indirect immunofluorescence assay (IFA) method. When fluorescence reaches +++, disperse the cells with trypsin, wash with 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 or rats with Hantavirus antigen inactivated by β-propiolactone.
5.1.4 Negative Serum
Serum from SPF mice or rats.
5.1.5 Enzyme Conjugate
Horseradish peroxidase-labeled goat or rabbit anti-mouse or anti-rat IgG antibody, used for detecting corresponding animal serum antibodies; Horseradish peroxidase-labeled Staphylococcal Protein A (SPA), used for detecting mouse serum antibodies.
5.1.6 Fluorescent Conjugate
Fluorescein isothiocyanate-labeled goat or rabbit anti-mouse or anti-rat IgG antibody, used for detecting corresponding animal serum antibodies.
5.1.7 Nucleic Acid Detection Reagents
5.1.7.1 DEPC-treated nuclease-free water.
5.1.7.2 Viral RNA extraction kit.
5.1.7.3 Real-time quantitative RT-PCR master mix (basal reaction solution).
5.1.7.4 Positive control: Inactivated animal tissue sample or cell culture containing Hantavirus.
5.1.7.5 Negative control: Tissue sample not containing Hantavirus.
5.1.7.6 Synthesize primers and probes according to the sequences in Table 1, and prepare as 10 μmol/L stock solutions. Store at -20°C.
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 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 14926.50-2001.
6.2 Use the IFA method for serological detection, operating according to GB/T 14926.52-2001.
6.3 Use the real-time quantitative RT-PCR method for Hantavirus nucleic acid detection, operating according to Appendix A.
7 Result Interpretation
