GB/T 14926.26-2026 Laboratory animal—Method for examination of Theiler's murine encephalomyelitis virus（TMEV） 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 14926.26-2026
Replaces GB/T 14926.26-2001
Laboratory animal - Method for examination of Theiler's murine encephalomyelitis virus（TMEV）
实验动物 小鼠脑脊髓炎病毒检测方法
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 Theiler's Murine Encephalomyelitis Virus
1 Scope
This document describes detection methods for Theiler's murine encephalomyelitis virus (TMEV).
This document applies to the detection of TMEV antibodies and nucleic acid in laboratory mice, 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
GB/T 14926.54 Laboratory animal – Haemagglutination inhibition test
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, TMEV antigen is used to detect TMEV antibodies in mouse serum. Alternatively, based on the principle that TMEV, under certain conditions, can agglutinate human type "O" erythrocytes, and this hemagglutination ability can be inhibited by specific antibodies, TMEV antibodies in mouse serum can be detected.
4.2 Principle of Nucleic Acid Detection
Specific primers and probes are designed and synthesized based on the conserved gene sequence of the TMEV untranslated region (UTR) to amplify target fragments. Real-time quantitative reverse transcription polymerase chain reaction (real-time quantitative RT-PCR) is used to detect whether TMEV 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 mice with TMEV (GDⅦ strain). After the onset of disease, aseptically collect brains, grind them, and prepare a 10% suspension. Centrifuge at 3000 r/min for 10 min, and use the supernatant to inoculate BHK21 cells. Allow adsorption for 1.5 h to 2 h, add maintenance medium, and culture for approximately 4 to 5 days. Harvest when cytopathic effect (CPE) reaches ++ to +++. 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 BHK21 cells, followed by low-speed centrifugation to remove cell debris.
5.1.2 Antigen Slides
Infect BHK21 cells with TMEV (GDⅦ strain) and culture for 4 to 5 days. When CPE reaches ++ to +++, disperse the cells with trypsin, wash with PBS, and prepare smears. Air dry at room temperature. Fix with cold acetone for 10 minutes. Store at -20°C.
5.1.3 Hemagglutinin
Intracerebrally inoculate TMEV (0.02 mL/mouse) into 14 to 21-day-old mice. After 5 to 7 days, aseptically collect brains, grind them, and prepare a 10% suspension. Freeze-thaw 2 to 3 times. Centrifuge at 1000 r/min for 10 min. Add an equal volume of 0.5% trypsin to the supernatant. Can be stored short-term at 4°C.
5.1.4 Positive Serum
Antiserum obtained by immunizing specific pathogen free (SPF) mice with TMEV virus antigen.
5.1.5 Negative Serum
Serum from SPF mice.
5.1.6 Enzyme Conjugate
Horseradish peroxidase-labeled goat or rabbit anti-mouse IgG antibody; or Horseradish peroxidase-labeled Staphylococcal Protein A (SPA).
5.1.7 Fluorescent Conjugate
Fluorescein isothiocyanate-labeled goat or rabbit anti-mouse IgG antibody.
5.1.8 Nucleic Acid Detection Reagents
5.1.8.1 DEPC-treated RNase-free water or commercially available RNase-free water.
5.1.8.2 Commercially available RNA extraction kit.
5.1.8.3 Real-time quantitative RT-PCR master mix: commercially available reagent.
5.1.8.4 Positive control: Tissue sample or cell culture containing TMEV virus nucleic acid.
5.1.8.5 Negative control: Tissue sample or cell culture not containing TMEV virus nucleic acid.
5.1.8.6 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 hemagglutination inhibition test (HAI) method for serological detection, operating according to GB/T 14926.54.
6.5 Use the real-time quantitative RT-PCR method for TMEV nucleic acid detection, operating according to Appendix A.
7 Result Interpretation
7.1 Interpretation of Antibody Detection Results
For positive results, retest using the same method or another method. If still positive, the result is interpreted as positive.
7.2 Interpretation of Nucleic Acid Detection Results
For positive or suspicious results, retest using the same method. If still positive or suspicious, the result is interpreted as positive.
8 Result Reporting
Issue a report based on the interpreted results.
