Establishment and preliminary verification of a highly sensitive nucleic acid extraction method for OBI detection

Abstract

Objective To establish and verify a new nucleic acid extraction method for OBI detection with large volume and high sensitivity, and apply it in the quantitative determination of OBI samples with low viral load. Methods The method for nucleic acid extraction with large volume was established based on the method of Roche nucleic acid detection kit. HBV standards were configured into 10 000 IU/mL, 1 000 IU/mL, 100 IU/mL, 10 IU/mL and 1 IU/mL respectively, and nucleic acid was extracted from the 10 mL standards by magnetic beads. CT values of each concentration were detected by fluorescence quantitative PCR and each concentration gradient was detected in parallel duplicates. The logarithm of virus concentration was taken as the X-axis and the average CT values of two tests were taken as the Y-axis to construct the fluorescence quantitative standard curve and regression equation. Three repeated experiments were conducted to verify the stability of the method. This method was used to extract nucleic acid from OBI samples with low viral load, and fluorescence quantification was performed. Results The amplification efficiency of fluorescence quantitative standard curves ranged from 90% to 105%, and the regression equation was greater than 0.99. The variation coefficients of variation of CT values were 0.63%, 0.78%, 1.52%, 1.36% and 0.78%, respectively. This method can extract nucleic acid from OBI samples with viral load of 1 IU/mL for quantification. Conclusion The detection limit of HBV nucleic acid quantitative detection system can reach 1 IU/mL, and it has strong stability and high sensitivity, which can be used for the quantitative detection of OBI with low viral load.

Keywords

• hbv
• nucleic acid extraction in large volume
• fluorescence quantitative pcr
• low viral load
• obi