GB/T 40140-2021 in English

Analysis of the core content of the standard

This standard specifies the systematic identification method of Grape axis blight pathogen (Alternaria alternata), integrating traditional morphology with modern molecular biology techniques to form a three-level identification system:

Detailed explanation of technical points

1. Key parameters of separation and culture

Double verification using PDA culture medium and PCA culture medium:

• Lighting conditions: 4200K color temperature lamp, 40cm away from the culture
• Temperature gradient: PDA 25℃ vs PCA 22℃
• Cultivation cycle: 3-4 days for initial screening → 7 days for morphological confirmation

2. Optimization of molecular detection system

Dual-track verification mechanism ensures accuracy:

Recommendations for the implementation of the standard

1. Laboratory configuration requirements

Essential equipment includes: biological microscope (400× and above), real-time fluorescence PCR instrument, and gel imaging system. Equipment calibration is required regularly.

2. Key points of quality control

• A positive control (ATCC67039) and a negative control (sterile water) must be set for each batch of experiments
• During DNA extraction, the A260/A280 ratio should be controlled at 1.7-1.9
• Morphological measurement requires statistical data of at least 30 conidia

3. Handling of difficult samples

For PCR gray zone results (35

1. Increase the amount of template DNA to 50-100ng
2. Use nested PCR to improve sensitivity
3. Combined with microscopic and ultrastructural observation

Analysis of standard evolution

Compared with historical methods, this standard has three major breakthroughs:

1. Etiology revision: The pathogen was confirmed to be Alternaria alternata rather than the previously misidentified A. viticola
2. Technology integration: Real-time fluorescence PCR was incorporated into the quarantine standard method for the first time
3. Parameter quantification: Precisely define the culture conditions (such as the lamp distance of 40cm) and the judgment threshold