GB/T 17494-2023 (English Version)

Standard No.: GB/T 17494-2023
Language: Chinese, Available in English version
Release Date: 2023
Published By: General Administration of Quality Supervision, Inspection and Quarantine of the People‘s Republic of China
Latest: GB/T 17494-2023
Replace By: GB/T 17494-2009

Scope: This document describes the methods of clinical diagnosis, real-time fluorescence PCR, virus isolation and identification, agar gel immunodiffusion test, indirect ELISA, competitive ELISA, and immunoblot test of EIA. This document is applicable to the diagnosis, quarantine, monitoring and epidemiological investigation of EIA.

Introduction

1. Standard Overview

GB/T 17494—2023 "Diagnostic Technology for Equine Infectious Anemia" is the latest revised technical standard for the diagnosis of equine infectious diseases in my country, replacing the old version GB/T 17494—2009. This standard mainly targets animal diseases caused by equine infectious anemia virus (EIAV), and regulates laboratory detection methods and biosafety requirements.

2. Background of Standard Revision

The revision of this standard is based on the following reasons:

Increase clinical diagnosis content and improve diagnostic process
Introduce more advanced nucleic acid detection technology (real-time fluorescence PCR)
Supplement virus isolation and identification methods
Optimize ELISA detection system

3. Comparison of Standard Frameworks

Chapter Content	Old Version (GB/T 17494—2009)	New Version (GB/T 17494—2023)
Scope	Only covers indirect ELISA detection technology	Comprehensively covers clinical diagnosis, nucleic acid detection, virus isolation and other methods
Laboratory biosafety	No clear regulations	Added GB 19489 requirements to strengthen biosafety measures
Diagnostic methods	Mainly indirect ELISA	New technologies such as real-time fluorescence PCR, virus isolation and identification

4. Major technical changes

New content:

Clinical diagnostic process (see Chapter 6)
Real-time fluorescence PCR detection method (see Chapter 8)
Virus isolation and identification technology (see Chapter 9)
Agar gel immunodiffusion test, competitive ELISA and immunoblotting test (see Chapters 10-13)

Revised content:

Optimize the indirect ELISA detection method (see Chapter 11)
Unify laboratory biosafety requirements (see Chapter 5)

5. Interpretation of laboratory diagnostic technology

5.1 Real-time fluorescence PCR detection

Real-time fluorescence PCR is a rapid and sensitive nucleic acid detection method that determines the presence of target nucleic acids based on real-time monitoring of changes in fluorescence signals during the PCR amplification process. This standard uses the probe method (see Table 1), which has the characteristics of high specificity and low detection limit.

Components	Volume/μL
2× Real-time PCR buffer	12.5
Upstream primer F1 (10μmol/L)	0.75
Downstream primer R1-3 (10 μmol/L)	0.5
Probe P1_rc (10 μmol/L)	0.8
Template DNA	2
Nuclease-free water	8.45
Total volume	25

5.2 Virus isolation and identification

Virus isolation is the process of obtaining EIAV virus through cell culture, which is mainly used for further research on virus strains and vaccine development. This standard recommends the use of a CO₂ cell culture incubator for culture and identification by real-time fluorescence PCR (see Chapter 9).

5.3 ELISA detection method

ELISA (enzyme-linked immunosorbent assay) is a commonly used antibody detection method. This standard covers the following ELISA techniques:

Indirect ELISA: used to detect EIAV-specific antibodies in serum
Competitive ELISA: determines the presence of antibodies by inhibition rate (see Formula 1)
Western blotting test: combined with SDS-PAGE technology, used for molecular weight detection of antigens

6. Implementation recommendations

6.1 Biosafety measures

Laboratory operations must strictly follow the requirements of GB 19489, especially the sample collection, storage and transportation links must comply with NY/T 541 specifications. It is recommended to use disposable blood collection needles and disposable blood collection tubes to avoid cross contamination.

6.2 Reagent preparation and quality control

All reagents should be prepared in accordance with the requirements of Appendix A, and it is recommended to calibrate instruments and equipment regularly, such as real-time fluorescence PCR instrument and enzyme reader.

6.3 Specimen collection and processing

For whole blood samples used for nucleic acid testing, it is recommended to use blood collection tubes containing heparin anticoagulant (see 7.2.2). Spleen tissue samples can be used for virus isolation experiments (see 7.3.3).

GB/T 17494-2023 Referenced Document

GB 19489 Laboratories.General requirements for biosafety
GB/T 6682 Water for analytical laboratory use.Specification and test methods
NY/T 541 Technical specifications for collection,storage and transportation of veterinary diagnostic specimens

GB/T 17494-2023 history

2023 GB/T 17494-2023 Diagnostic Technology for Equine Infectious Anemia
2009 GB/T 17494-2009 Diagnostic techniques of indirect ELISA technique for equine infectious anemia disease
1998 GB/T 17494-1998 Rules of indirect ELISA technique for equine infectious anemia disease