SC/T 7231-2019 in English

This standard establishes a complete technical system from sampling to molecular detection for shellfish diseases caused by Marteilia refringens. It focuses on standardizing the 413bp target sequence amplification process of PCR detection, and explicitly requires the use of primers Pr4/Pr5 combination.

1. Sample processing

Different tissues need to be collected according to the growth stage of shellfish: viscera mass from juvenile shellfish, gills/intestines/digestive glands from adult shellfish. Storage temperature must be ≤-20℃.

2. DNA extraction

The phenol-chloroform three-step extraction method was used, with the following key parameters: final proteinase K concentration of 100 μg/mL, digestion at 50°C for 3 hours, and DNA precipitation with ammonium acetate.

3. PCR amplification

The reaction system contained 2.5 mM MgCl₂, 0.4 μM primers, and cycle parameters: 94°C pre-denaturation for 5 min → 35 cycles (94°C for 30 s → 55°C for 30 s → 72°C for 30 s) → 72°C extension for 7 min.

• Monitoring in epidemic areas: give priority to the combined use of PCR and tissue printing method
• Import quarantine: electron microscopy verification is required (especially for shellfish from the Mediterranean/North Atlantic)
• Laboratory quality control: each batch must have a positive control (ATCC preserved strain) and a blank control

This standard is formulated based on the requirements of the OIE Aquatic Animal Health Code, and reflects my country's needs for the prevention and control of exotic shellfish diseases. The 2019 version has been upgraded mainly in the following aspects:

1. Primer specificity validation data
2. Compatibility description of commercial kits
3. Step-by-step determination process for diagnosis in non-epidemic areas