SN/T 5671-2024 in English

Analysis of Standard Technical Framework

Key Points of Core Detection Technology

1. Instrument Configuration Requirements

The standard clearly requires the use of Class II biosafety cabinets, real-time fluorescence PCR amplifiers and digital PCR systems (including micro-reaction system generators and detectors), of which the digital PCR system must ensure that the micro-reaction system generation stability is ≥60%.

2. Primer probe design

A dual detection strategy is adopted:
- PCV2: HEX fluorescent labeled probe, amplified fragment 87/118bp
- PCV3: FAM fluorescent labeled probe, amplified fragment 76/86bp
The internal reference gene (IC) uses the Cy5 channel to ensure the effectiveness of the reaction.

Quality control system

1. Three-level control setting

• Blank control: Nuclease-free water instead of template
• Negative control: Non-infected tissue/blood
• Positive control: Standard substance containing PCV2/3

2. Calculation of digital PCR recovery rate

The detection efficiency was evaluated by exogenous process control (EPC), and the recovery rate was required to be >1%. The calculation formula is:
B=(CEPC×A×V1)/(CO×V2)
Where A is the dilution multiple and V1/V2 are volume parameters.

Implementation suggestions

1. Sample pre-treatment optimization

Tissue samples are recommended to be homogenized with 1:5 (m/v) physiological saline and repeatedly frozen and thawed 3 times to improve the efficiency of nucleic acid release. Blood samples need to be added with 10μL EPC for process monitoring.

2. Critical value treatment plan

When 37 - Replace the reagent batch for retest
- Use digital PCR verification
- Combine epidemiological data for comprehensive judgment

3. Biosafety management

Strictly implement the requirements of GB19489, waste needs to be autoclaved at 121℃ for 30 minutes, and experimental personnel should obtain the second-level biosafety operation qualification.