GB/T 35900.2-2018 in English

Background and Significance of the Standard

GB/T 35900 "Animal Influenza Detection" is an important national standard proposed by the Ministry of Agriculture of the People's Republic of China, which aims to standardize the detection methods of animal influenza viruses. This part (Part 2) is specifically for the fluorescent RT-PCR detection method of H3 subtype influenza virus nucleic acid.

The background of the formulation of this standard mainly includes:

• Ensure animal health and public health safety
• Unify the national animal influenza detection technical requirements
• Improve laboratory detection efficiency and accuracy

Standardized laboratory settings and biosafety

According to GB/T 19438.1-2004, the laboratory must meet the following conditions:

• The biosafety level requirements must comply with GB19489 standards
• Equipped with dedicated testing areas (sample preparation area, reaction mixture preparation area, amplification area)
• Strictly control personnel training and operating specifications

Reagent and instrument requirements

According to the standard, the main reagents required include:

• TRIzol: used for RNA extraction
• DEPC water: to remove nuclease contamination
• Fluorescent PCR reaction solution: containing MGB modified probe

Main instruments and equipment include:

• Fluorescent PCR detector (with matching reaction tubes)
• High-speed desktop refrigerated centrifuge (≥12000 r/min)
• Tissue homogenizer: for sample processing

Sample collection and pretreatment process

The following key steps should be noted in sample collection:

• Swab samples:Throat, cloaca or nasal secretions, stored in PBS solution
• Tissue samples:Lung or tracheal tissue, treated with PBS solution
• Sample transportation: Send to the laboratory within 24 hours. Long-term storage requires freezing at -70℃

Detailed explanation of fluorescent RT-PCR detection method

The specific operation steps are as follows:

1. Sample nucleic acid extraction: Use TRIzol lysis method to separate RNA and remove impurities
2. Reagent preparation: Prepare fluorescent RT-PCR reaction solution according to the formula in Table B.2
3. Amplification and detection: Use fluorescence PCR detector and set the parameters as follows:
   • Reverse transcription: 42℃/30min
   • Pre-denaturation: 94℃/3min
   • Cycling parameters: 94℃/15s, 50℃/10s, 60℃/35s (40 cycles)

Result determination and quality control

Determine the result based on the Ct value:

• Negative:No amplification curve and Ct value>30
• Positive:Ct value ≤30, with a typical amplification curve
• Re-test principles:If the Ct value>30 but there is an amplification curve, re-testing is required

Implementation suggestions and precautions

To ensure the detection effect, it is recommended that:

• Laboratory personnel must undergo professional training
• Strictly control the reagent preparation environment to avoid contamination
• Regularly calibrate instruments and perform performance verification
• Establish a negative and positive control system to ensure the effectiveness of the experiment