Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Nucleic acid (NA) isolation is essential for genetic analysis and biosensor development.
• Contaminants in purified NA, such as salts and organic compounds, reduce analytical sensitivity, especially in DNA biosensors.
• Optimizing viral NA purification is critical to mitigate these inhibitory effects.

Purpose of the Study:

• To optimize factors affecting viral nucleic acid purification.
• To enhance the analytical sensitivity of DNA biosensors by improving NA purity.
• To develop a semi-automated system for simultaneous viral NA extraction.

Main Methods:

• Investigated the impact of guanidinium hydrochloride concentration, lysis buffer pH, carrier RNA, and ethanol addition on NA yield and quality.
• Compared elution efficiency using DNase/RNase-free water, TE buffer, and PCR buffer.
• Optimized parameters for an automated NA extractor for simultaneous HBV DNA and HCV RNA extraction.

Main Results:

• A guanidinium hydrochloride concentration of 6 M was found critical for NA isolation.
• Optimal NA yield and quality were observed at a lysis buffer pH of 4–5.
• Carrier RNA was indispensable for viral genome isolation and eluted best in DNase/RNase-free water or TE buffer.
• Ethanol addition (1:1 ratio) significantly improved NA recovery.
• The automated system successfully extracted and amplified HBV DNA and HCV RNA, demonstrating reliable performance.

Conclusions:

• Established an optimized protocol for viral nucleic acid purification, critical for downstream genetic applications and biosensor development.
• Demonstrated the effectiveness of carrier RNA and specific buffer conditions for maximizing NA yield and purity.
• Developed and validated a semi-automated system for high-throughput, simultaneous detection of multiple viral nucleic acids.