GB/T 14926.22-2026 Laboratory animal—Method for examination of Mouse hepatitis virus（MHV） 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 14926.22-2026
Replaces GB/T 14926.22-2001
Laboratory animal - Method for examination of Mouse hepatitis virus（MHV）
实验动物 小鼠肝炎病毒检测方法
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 Reagents and Equipment
6 Detection Methods
7 Result Interpretation
8 Result Reporting
Laboratory Animals — Detection Method for Mouse Hepatitis Virus
1 Scope
This document describes detection methods for Mouse hepatitis virus (MHV).
This document applies to the detection of MHV in mice, or in samples originating from experimental inocula or 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 14926.42 Laboratory animal – Bacteriological examination – Collection of specimens
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, MHV antigen is used to detect MHV antibodies in mouse serum.
4.2 Principle of Nucleic Acid Detection
Common MHV strains in laboratory animals possess unique genomic nucleic acid sequences, which can be identified by nucleic acid amplification techniques such as polymerase chain reaction (PCR).
5 Reagents and Equipment
5.1 Reagents
5.1.1 Antibody Detection Reagents
5.1.1.1 Enzyme-linked immunosorbent assay (ELISA) Specific Antigen
Infect DBT or L929 cells with MHV virus (including strains MHV1, MHV3, MHV-A59, MHV-JHM). Harvest 2 to 4 days post-infection when cytopathic effect (CPE) reaches +++, ++++. Freeze-thaw 3 times or sonicate. Remove cell debris by low-speed centrifugation. Concentrate the supernatant by ultracentrifugation to prepare the ELISA antigen.
5.1.1.2 ELISA Normal Antigen
Supernatant obtained by freeze-thawing and disrupting uninfected DBT or L929 cells, followed by low-speed centrifugation to remove cell debris.
5.1.1.3 Antigen Slides
Infect DBT or L929 cells with MHV virus. 1 to 2 days post-infection, when CPE reaches ++, +++, disperse the cells with trypsin, wash with phosphate buffer saline (PBS), and prepare smears. Air dry at room temperature. Fix with cold acetone for 10 minutes. Store at -20°C.
5.1.1.4 Positive Serum
Antiserum obtained by immunizing specific pathogen free (SPF) or germ-free mice with MHV virus antigen.
5.1.1.5 Negative Serum
Serum from SPF or germ-free mice.
5.1.1.6 Enzyme Conjugate
Horseradish peroxidase-labeled goat or rabbit anti-mouse IgG antibody; or Horseradish peroxidase-labeled Staphylococcal Protein A (SPA).
5.1.1.7 Fluorescent Conjugate
Fluorescein isothiocyanate-labeled goat or rabbit anti-mouse IgG antibody.
5.1.2 Nucleic Acid Detection Reagents
5.1.2.1 Primers and probes for real-time quantitative PCR amplification are shown in Table 1.
5.1.2.2 Prepare other reagents according to Appendix A.
5.2 Equipment
5.2.1 Antibody Detection Equipment
5.2.1.1 Microplate reader.
5.2.1.2 Fluorescence microscope.
5.2.1.3 Ordinary light microscope.
5.2.1.4 37°C incubator or water bath.
5.2.2 Nucleic Acid Detection Equipment
Prepare equipment related to nucleic acid detection methods according to Appendix A.
6 Detection Methods
6.1 Sample Collection and Processing
6.1.1 Collect serum according to the method in GB/T 14926.42.
6.1.2 Collect organ tissues, cecal contents or feces, experimental inocula, and environmental samples from laboratory animal facilities according to Appendix A.
6.2 Sample Detection Methods
6.2.1 Use the ELISA method for serological detection, operating according to GB/T 14926.50.
6.2.2 Use the indirect immunofluorescence assay (IFA) method for serological detection, operating according to GB/T 14926.52.
6.2.3 Use the immunoenzyme assay (IEA) method for serological detection, operating according to GB/T 14926.51.
6.2.4 Use the real-time quantitative PCR method for MHV nucleic acid detection, operating according to Appendix A.
7 Result Interpretation
7.1 ELISA/IFA/IEA
For positive results, retest using the same method or another serological method. If still positive, the result is interpreted as positive.
7.2 Real-time Quantitative PCRtis virus(MHV)
