GB/T 14926.24-2026 Laboratory animal—Method for examination of pneumonia virus of mice (PVM) 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 14926.24-2026
Replaces GB/T 14926.24-2001
Laboratory animal - Method for examination of pneumonia virus of mice (PVM)
实验动物 小鼠肺炎病毒检测方法
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 Pneumonia Virus of Mice
1 Scope
This document describes detection methods for Pneumonia virus of mice (PVM).
This document applies to the detection of PVM in mice, rats, hamsters, guinea pigs, as well as in experimental inocula 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 14926.42 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, PVM antigen is used to detect PVM antibodies in the serum of mice, rats, hamsters, and guinea pigs.
4.2 Principle of Nucleic Acid Detection
Based on molecular biology principles, the unique genomic nucleic acid sequences of Pneumonia virus of mice can be identified by nucleic acid amplification techniques such as polymerase chain reaction (PCR), thereby obtaining the target gene or fluorescent signal for detection.
5 Reagents and Equipment
5.1 Reagents
5.1.1 Enzyme-linked Immunosorbent Assay (ELISA) Antigen
5.1.1.1 Specific Antigen
After infecting mice with PVM and awaiting the onset of disease, collect the lungs, grind them, and prepare a 10% suspension. Centrifuge at 3000 r/min for 10 min, and use the supernatant to infect BHK21 cells. Allow adsorption for 1.5 h to 2 h, add maintenance medium, and culture for 10 to 14 days. Harvest 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 Normal Antigen
Supernatant obtained by freeze-thawing and disrupting uninfected BHK21 cells, followed by low-speed centrifugation to remove cell debris.
5.1.2 Antigen Slides
Infect BHK21 cells with PVM and culture for 5 to 7 days. When CPE reaches ++ to +++, disperse the cells with trypsin, wash with phosphate-buffered saline (PBS), and prepare smears. Air dry at room temperature. 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 PVM.
5.1.4 Negative Serum
Serum from SPF mice, rats, guinea pigs, and hamsters.
5.1.5 Enzyme Conjugate
Horseradish peroxidase, etc. 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, etc. labeled goat or rabbit anti-mouse, rat, guinea pig, hamster IgG antibodies, used for detecting corresponding animal serum antibodies.
5.1.7 Primers and Probes
Synthesize primers and probes according to the sequences in Table 1. Prepare primers and probes as 10 μmol/L stock solutions using RNase-free deionized water. 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 quantitative PCR instrument.
6 Detection Methods
6.1 Collect serum according to GB/T 14926.42.
6.2 Collect organ tissues, cecal contents or feces, experimental inocula, and environmental samples from laboratory animal facilities according to Appendix A.
6.3 Use the ELISA method for serological detection, operating according to GB/T 14926.50.
6.4 Use the indirect immunofluorescence assay (IFA) method for serological detection, operating according to GB/T 14926.52.
6.5 Use the immunoenzyme assay (IEA) method for serological detection, operating according to GB/T 14926.51.
6.6 Use the real-time quantitative RT-PCR method for nucleic acid detection, operating according to Appendix A.
7 Result Interpretation
7.1 Serological Detection
For positive results, retest using the same serological method or another serological method. If still positive, the result is interpreted as PVM antibody positive.
7.2 Nucleic Acid Detection
For positive or suspicious results, retest using the same method. If still positive or suspicious, the result is interpreted as positive.
