GB/T 36847-2018 in English

1. Standard Overview

This standard specifies the isolation, culture, immunology and molecular biology detection and identification methods of orchid brown spot pathogen, which is applicable to the quarantine and identification of orchid brown spot pathogen in orchid plants and the field investigation and monitoring of the disease.

2. Detection Principle

• Immunological detection: based on the specific reaction between pathogens and antibodies.
• Molecular biological detection: PCR amplification and electrophoresis analysis based on the specific DNA sequence of pathogens.
• Isolation, culture and identification: separation and purification through King's B medium, combined with Biolog system and pathogenicity determination for confirmation.

3. Laboratory testing process

1. Sample preparation: Disinfect the tissue at the junction of the diseased and healthy tissues and crush them to make a suspension.
2. Immunological detection: Use commercial ELISA kits for detection.
3. Molecular biological detection: After extracting DNA, perform PCR amplification and electrophoresis analysis or real-time fluorescence PCR detection.
4. Isolation and culture identification: Streak on King's B medium to isolate and confirm the strain with Biolog system and pathogenicity determination.

4. Comparison of standard frameworks

5. Implementation recommendations

• PCR testing should be preferred as the primary screening method because of its high sensitivity and good specificity.
• For positive samples, final confirmation should be carried out by combining isolation culture and pathogenicity determination.
• Strengthen laboratory staff training to ensure standardized operations.
• Establish a data sharing mechanism to facilitate subsequent research and supervision.