GB/T 38488-2021 in English

Analysis of standard technical framework

Detailed explanation of key technical principles

1. Innovation of the molecular motor method

After the ε subunit antibody-streptavidin-biotin-nucleic acid probe complex (see standard Appendix A) binds to the target gene, it causes the ATP synthase rotation speed to change, and the DHPE fluorescent probe is used to detect changes in the amount of proton transmembrane transport. This method breaks through the limitations of traditional PCR technology and realizes non-amplification direct detection.

2. Advantages of near-infrared immunochromatography

775nm/795nm near-infrared fluorescent labeling (Appendix C of the standard) is used to effectively avoid the interference of food matrix autofluorescence. The threshold of the fluorescence intensity ratio between the test line and the quality control line is set to negative control value + 3SD, which significantly improves the signal-to-noise ratio.

3. Key points of target region sequencing method quality control

The sample barcode is required not to be reused within 30 days (Standard 6.4.3.3), and the data reliability is ensured by the dual indicators of C1≥30×10³ and C2≥0.3×10⁹. Positive is determined when the number of labeled sites is ≥5.

Recommendations for the implementation of the standard

1. Method selection: Near-infrared immunochromatography is preferred for high-throughput screening. For difficult samples, the molecular motor method combined with sequencing is recommended
2. Result verification: All positive results must be confirmed by the classical method according to Appendix B
3. Staff training: The molecular motor method requires special training on rotor speed calibration technology
4. Equipment management: Near-infrared scanners must be calibrated regularly with standard fluorescent microspheres

Technology evolution analysis

This standard incorporates molecular motor technology into the national standard system for the first time, shortening the detection cycle from 3-5 days to 4-8 hours compared to traditional culture methods. The detection limit of near-infrared immunochromatography is 10 times higher than that of ELISA, and the sequencing throughput of the target area reaches 0.5Gb/sample, which means that microbial detection is developing in the direction of trace, rapid and multi-target.