GB/T 14926.20-2026 Labratory animal—Method for examination of Ectromelia virus(ECT) 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 14926.20-2026
Replaces GB/T 14926.20-2001
Laboratory animal - Method for examination of Ectromelia virus(ECT)
实验动物 鼠痘病毒检测方法
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 Ectromelia Virus
1 Scope
This document describes detection methods for Ectromelia virus (ECT).
This document applies to the detection of Ectromelia virus in laboratory animals, or the detection of Ectromelia virus 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 14926.50 Laboratory animal – Enzyme-linked immunosorbent assay
GB/T 14926.51 Laboratory animal – Immunoenzyme assay
GB/T 14926.52 Laboratory animal – Immunofluorescence assay
GB/T 14926.55 Laboratory animal – Immunohistochemistry method
GB 19489 Laboratory – General requirements for biosafety
GB/T 19495.2 Detection of genetically modified organisms and derived products – General requirements for laboratories
3 Terms and Definitions
No terms and definitions are required for this document.
4 Principle
Enzyme-linked immunosorbent assay (ELISA) / Immunoenzyme assay (IEA): ECT antigen is used to detect ECT antibodies in mouse serum. The resulting antigen-antibody complex binds to a corresponding enzyme-labeled secondary antibody. Under enzymatic catalysis, the substrate reacts, producing a colored substance. The intensity of the color reaction is proportional to the amount of ECT antibody present in the test serum.
Immunofluorescence assay (IFA): ECT antigen is used to detect ECT antibodies in mouse serum. The resulting antigen-antibody complex binds to a corresponding fluorescein-labeled secondary antibody. Under ultraviolet or blue-violet light excitation, visible fluorescence is emitted. The presence and intensity of fluorescence are observed under a fluorescence microscope to determine the result.
Immunohistochemistry (IH): Known ECT-specific antibodies react with the target antigen in the specimen. If the target antigen corresponds to the specific antibody, an antigen-antibody complex forms. This complex retains its antigenic activity and can bind to a corresponding secondary antibody-enzyme conjugate. Upon exposure to the enzyme substrate, a color reaction occurs. The result is determined under a light microscope based on the color reaction.
Nucleic Acid Detection: Based on conserved gene sequences of Ectromelia virus, a pair of specific primers and a specific probe sequence are designed and synthesized. DNA is extracted from the sample and amplified using real-time quantitative PCR. The detection result determines whether viral nucleic acid components are present in the sample.
5 Main Reagents and Equipment
5.1 Main Reagents
5.1.1 ELISA Antigen
5.1.1.1 Specific Antigen
Infect BHK21 cells with ECT. 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 ECT. 2 to 3 days post-infection, 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 Positive Serum
Antiserum obtained by immunizing specific pathogen free (SPF) mice with inactivated ECT antigen.
5.1.4 Negative Serum
Serum from SPF mice.
5.1.5 Enzyme Conjugate
Horseradish peroxidase-labeled goat or rabbit anti-mouse IgG antibody; or Horseradish peroxidase-labeled Staphylococcal Protein A (SPA).
5.1.6 Fluorescent Conjugate
Fluorescein isothiocyanate-labeled goat or rabbit anti-mouse IgG antibody.
5.1.7 Primers and Probes
Synthesize primers and probes according to the sequences in Table 1. Prepare primers and probes as 20 μmol/L stock solutions using DNase-free deionized water. Store at -20°C.
5.2 Instruments and 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 Paraffin microtome or cryostat (freezing microtome).
5.2.6 Real-time quantitative PCR instrument.
6 Detection Methods
6.1 Experimental operations and handling shall be carried out by qualified personnel according to the provisions of GB 19489.
6.2 General technical requirements for laboratories and basic requirements for test quality control shall comply with GB/T 19495.2.
6.3 Sampling and sample handling shall be carried out according to Appendix A. The sampling process must ensure no cross-contamination of samples.
6.4 Use the ELISA method for serological detection, operating according to GB/T 14926.50.
6.5 Use the IFA method for serological detection, operating according to GB/T 14926.52.
6.6 Use the IEA method for serological detection, operating according to GB/T 14926.51.
6.7 Use the IH method for viral antigen detection, operating according to GB/T 14926.55.
6.8 Use the real-time quantitative PCR method for ECT 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; otherwise, it is interpreted as negative.
7.2 IH
If the background of the control slide is clear, with no non-specific staining, and the target in the test specimen stains yellow to brownish-yellow, the result is interpreted as positive; otherwise, it is interpreted as negative.
7.3 Real-time Quantitative PCR
For positive or suspicious results, retest using the same method. If still positive or suspicious, the result is interpreted as positive; otherwise, it is interpreted as negative.
8 Result Reporting
Issue a report based on the interpreted results.
