GB/T 38740-2020 in English

Analysis of standard technical framework

Key technical points

1. Probe design principle

A TaqMan probe labeled with FAM-BHQ1 (sequence 5′-AGCAGGCGTTGAGCAACCTAGCCCGA-3′) was used. When the probe was cut by the Taq enzyme, the fluorescence signal was exponentially related to the viral RNA content. The critical value setting followed the 3σ principle, and the baseline noise was controlled at ≤5% of the fluorescence intensity.

2. Sample handling specifications

• Blood samples: Need to be collected using sterile anticoagulant tubes and stored at -70℃ to avoid repeated freezing and thawing
• Tissue samples: Liver/spleen need to be homogenized with PBS at a ratio of 1:5 (w/v)
• RNA extraction: Trizol method is recommended, with a yield of ≥80%

Implementation suggestions

1. Quality control plan

Each batch of tests should include:

1. Positive control (in vitro transcribed RNA, Ct≤28)
2. Negative tissue control
3. DEPC water blank control

2. Common problem handling

Standard Evolution Analysis

This standard establishes the monkey Marburg virus detection system in my country for the first time. Compared with ISO/IEC 17025:2017, it adds:

• Freeze-thaw number limit (≤3 times)
• High-speed refrigerated centrifuge parameter requirements (≥12000rpm)
• Clearly define the gray area judgment rule of 28＜Ct≤32