Is the nucleic acid extraction efficiency for low-load pathogens suboptimal? Are there extraction kits suitable for trace samples?

Poor extraction of low-load samples is not necessarily a kit problem; pre-processing and elution volume also have a major impact. It is recommended to use pseudovirus with a known copy number or quantitative quality control for spike-in recovery, run 3 replicates continuously across at least 3 batches, and examine Ct value variation. For cerebrospinal fluid with low protein and small sample volume, an internal standard or RNA carrier can be added to reduce adsorption, but the amount added must be fixed, otherwise it will interfere with quantification. If the recovery rate for a 200 μL sample is insufficient, a large-volume extraction plus concentration approach can be evaluated, but costs may increase. Before switching methods, take several low-concentration positive samples and run column extraction and magnetic bead method controls in parallel; do not judge based only on concentration values.

For sample extraction within a budget of 20 yuan, domestic magnetic bead-based methods can usually be negotiated down, but be sure to include deep-well plates, magnetic sleeves, pipette tips, and elution buffer in the calculation; imported reagents often tend to exceed the budget. It is recommended to request long-term supply pricing and batch-specific COAs from the manufacturer, and to first trial three different lot numbers. Pay close attention to whether magnetic bead residue remains after elution, as residue can inhibit amplification and cause Ct value fluctuations. In our laboratory, we previously used domestic magnetic beads for plasma DNA extraction with a 50 μL elution volume, and the Ct values were more stable than column-based extraction, with the overall cost per sample controllable within the low teens. Before switching, it is best to run a parallel comparison with a batch of weakly positive samples to confirm whether inventory and lead times are stable.

For low-load samples such as blood and cerebrospinal fluid, do not continue using conventional column-based extraction. Instead, look for magnetic bead reagents specifically designed for trace or cell-free nucleic acids, which usually contain carrier RNA or co-precipitation agents to reduce tube-wall adsorption of low-concentration nucleic acids. In terms of brands, you can test both imported and domestic options, such as Thermo Fisher, Qiagen, as well as domestic Tianlong, Sansure, and Vazyme. Request small packaging and evaluate recovery using known weakly positive samples. During testing, fix the elution volume and compare the Ct value difference before and after extraction, which is more practical than simply looking at the extracted concentration.