GB/T 14926.8-2026 Laboratory animal—Method for examination of Pneumocystis sp. 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 14926.8-2026
Replaces GB/T 14926.8-2001
Laboratory animal - Method for examination of Pneumocystis sp.
实验动物 支原体检测方法
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 Culture Media and Reagents
6 Main Equipment
7 Detection Methods
8 Result Interpretation
9 Result Reporting
Laboratory Animals — Detection Methods for Mycoplasma
1 Scope
This document describes detection methods for Mycoplasma in laboratory animals.
This document applies to the detection of Mycoplasma in laboratory animals such as mice and rats, as well as in laboratory animal 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 14926.42-2001 Laboratory animal – Bacteriological examination – Collection of specimens
GB/T 14926.43 Laboratory animal – Bacteriological examination – Staining, media and reagents
GB/T 14926.50 Laboratory animal – Enzyme-linked immunosorbent assay
Pharmacopoeia of the People's Republic of China
3 Terms and Definitions
No terms and definitions are required for this document.
4 Principle
Mycoplasma can form characteristic colonies on culture media, which stain a distinctive blue with Dienes stain. When detecting serum antibodies using enzyme-linked immunosorbent assay (ELISA), the antigen-antibody-enzyme conjugate complex catalyzes a chromogenic reaction; the color intensity is proportional to the antibody concentration. By designing primers and probes targeting conserved genes of Mycoplasma, the presence of nucleic acids can be determined through specific DNA fragment amplification via polymerase chain reaction (PCR).
5 Culture Media and Reagents
5.1 Semi-fluid Mycoplasma medium.
5.2 Liquid Mycoplasma medium.
5.3 Solid Mycoplasma medium.
5.4 Dienes stain.
5.5 Prepare ELISA antigen according to the following procedure:
a) Mycoplasma culture: Inoculate standard strains of Mycoplasma pulmonis, Mycoplasma arthritidis, and Mycoplasma neurolyticum into liquid Mycoplasma medium. Incubate at 36°C with shaking for 2 to 3 days.
b) Centrifuge the clearly turbid culture at 8000 r/min for 30 min. Wash the pellet three times with phosphate-buffered saline (PBS) under the same conditions.
c) Resuspend the pellet in PBS, disrupt by sonication, and use the supernatant as the ELISA antigen.
5.6 Enzyme conjugates include two types: Horseradish peroxidase, alkaline phosphatase, etc. labeled goat anti-mouse/rat (or rabbit anti-mouse/rat) IgG antibodies, used for detecting corresponding animal serum antibodies; or the aforementioned enzyme-labeled Staphylococcal Protein A (SPA), used for detecting mouse serum antibodies.
5.7 Positive serum is antiserum obtained by immunizing specific pathogen free (SPF) mice and rats with Mycoplasma antigen.
5.8 Negative serum is serum from SPF mice and rats free from Mycoplasma infection.
Prepare other reagent solutions according to the provisions of GB/T 14926.43.
5.9 Synthesize primers and probes according to the sequences in Tables 1, 2, and 3. Prepare primers and probes as 10 μmol/L stock solutions using nuclease-free water. Store at -20°C or below.
6 Main Equipment
6.1 General incubator.
6.2 Stereo microscope.
6.3 Vortex mixer.
6.4 Constant temperature water bath.
6.5 High-speed centrifuge.
6.6 Ultrasonic cell disruptor.
6.7 Microplate reader.
6.8 PCR instrument, Real-time quantitative PCR instrument.
6.9 Electrophoresis apparatus.
6.10 Gel imaging and analysis system.
7 Detection Methods
7.1 Sample Collection and Processing
7.1.1 Respiratory Tract Samples
Take approximately 5 mm to 10 mm of tissue including the pharynx and the lower trachea, and place it into a test tube containing 0.6 mL to 0.7 mL of yeast infusion broth. Vortex for 30 seconds to prepare an eluate.
7.1.2 Serum Samples
Proceed according to the sampling method specified in Chapter 4 of GB/T 14926.42-2001.
7.1.3 Other Samples
Collect nasopharyngeal swabs, inflamed joints, upper respiratory tract organs/tissues (nose, pharynx, larynx), lungs, experimental animal inocula, and environmental samples from laboratory animal facilities according to Appendix A.
7.2 Isolation Culture Method
7.2.1 Detection Procedure
The detection procedure is shown in Figure 1.
7.2.2 Operating Steps
7.2.2.1 Isolation Culture
Inoculate the samples collected in 7.1.1 according to the method in Chapter 4 of GB/T 14926.42-2001, then incubate in a (36 ± 1)°C incubator.
7.2.2.2 Identification
