Abstract
Objective To establish a method of nucleic acid extraction and enrichment based on magnetic nanoparticle as medium for elevating the analytical sensitivity of domestic HBV real-time PCR kit and detection of the trace amount HBV DNA. Methods After receiving antiviral treatment, the serum samples of 50 hepatitis B patients with HBV DNA concentration ≤1×104 IU/ml were collected. The WHO HBV DNA calibrator was used as the standard material. Nanometer magnetic beads were used to adsorb and enrich the HBV nucleic acid and increase the concentration of the extracted HBV nucleic acid template. Compared with Roche HBV DNA detection reagent and four domestic reagent with conventional nucleic acid extraction and detection method, the improvement effect of this method on domestic nucleic acid detection reagent was evaluated. Results After application of nanometer magnetic extraction method to domestic regent, the analytical sensitivities of the domestic reagent reached 10 and 50 IU/ml, respectively,which was about the same detection level to 12 IU/ml of the imported Roche reagent. The positive rates of the detection of serum trace amount HBV DNA of hepatitis B patients with four kinds of domestic extraction reagent were 64% ( 32 ), 56% ( 28 ), 62% ( 31 ) and 58% (29), respectively. There were significant statistical differences between Roche reagent and four domestic extraction reagent kits(x2 = 7. 895, 12. 698,9. 013 and 11. 416 ,P ＜0. 05 ). With nanometer magnetic extraction method combined with domestic reagent kits, the detection rates were 88% (44), 88% (44), 88% (44) and 86% (43) ,respectively. There was no significant difference compared with the imported Roche reagent (x2 = 0. 000, 0. 000, 0. 000 and 0. 088,P ＞0. 05). Moreover, when the HBV nucleic acid concentration was 101-103 IU/ml, the logarithm value of viral nucleic acid concentration was in reverse correlation to Ct value, but the correlation decreased in the concentration range of 103-106 IU/ml. Conclusions The nucleic acid extraction method based on magnetic nanoparticle as medium can significantly improve the analytical sensitivity of domestic HBV DNA detection reagent, which can be used to monitor the trace amounts HBV DNA in the sera of the hepatitis B patients. Key words: Hepatitis B virus; Magnetic nanoparticle; Nucleic acid test; Analysis sensitivity
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