Diagnostic Dilemma: False Signals from Nucleic Acid Residues In molecular biology research and diagnostic applications, pathogens are evolving in complex ways, often leaving patients undiagnosed due to unclear pathogens. Rapid and accurate detection is crucial, but background nucleic acid residues have become a key factor impacting the accuracy and reliability of test results.

When detecting pathogens such as respiratory viruses, gut microbiota, and bloodstream infections using techniques like PCR, tNGS, and mNGS, nucleic acid contamination can obscure low-abundance target nucleic acids or appear alongside them, affecting detection sensitivity or causing false positives. This can mislead diagnostics and treatment. In diagnostic reagent development, excessive nucleic acid residues in enzyme raw materials can reduce amplification specificity and accuracy, impacting results and analysis.

It has been shown that ultra-clean enzyme raw materials, with host cell DNA (HCD) as low as ≤ 0.001 copies/U or even undetectable levels, are essential to meet the stringent performance requirements of in vitro diagnostic reagents.

Purification Pathway: Guide to Developing Ultra-Clean Molecular Enzymes

Completely removing nucleic acid residues is challenging. Host nucleic acid molecules are stable, often binding with proteins and remaining in the final product. Furthermore, environmental and procedural contamination can add nucleic acids. To eliminate residues, both external and internal sources of contamination must be addressed, using advanced techniques and stringent quality controls to achieve ultra-clean enzyme development.

External Defense: Removing External Nucleic Acid Contamination

To minimize external nucleic acid contamination, a well-planned laboratory or production layout is essential, with distinct zones to reduce cross-contamination. Regular use of disinfectants like 70%-80% ethanol or sodium hypochlorite on surfaces and equipment can help control environmental nucleic acids. Sodium hypochlorite is particularly effective against aerosols.

Following GMP quality standards during production, using gloves, masks, lab coats, and avoiding direct contact with materials, along with tools like pipettes and closed systems, can reduce manual contamination. For example, consumables like glycerol and pipette tips have shown varying nucleic acid residues. Single-use consumables can significantly reduce contamination risks.

Internal Purification: Strategies for Removing Internal Nucleic Acid Residues

Effective processes often remove a substantial amount of nucleic acids during initial sample preparation, with further removal in chromatography steps, ensuring consistently low nucleic acid residue levels in the final product.

1.Preliminary Nucleic Acid Purification: After cell lysis, nucleic acids can be removed by precipitation, utilizing their negative charge to bind with positively charged substances like polyethyleneimine (PEI), streptomycin sulfate, or protamine sulfate. Enzymatic digestion can also remove nucleic acid contamination, with optimal conditions for enzymes like Benzonase or DNase I determined through DOE analysis.

2.Multiple Chromatography Purifications: Chromatography methods like heparin, anion exchange, gel filtration, and hydrophobic interaction chromatography (HIC) effectively remove nucleic acid contaminants, leveraging the unique properties of nucleic acids.

• Heparin Chromatography: Used in biological products like Protein C and fibrinogen, heparin chromatography significantly reduces HCD by mimicking nucleic acid interactions.