CAJ | 학술논문

建立一种基于纳米磁珠为介质的核酸提取和富集方法,提高国产HBV核酸检测试剂的分析灵敏度,用于痕量HBV DNA的测定.方法选择经抗病毒治疗HBV DNA浓度≤1×104 IU/ml的乙型肝炎患者血清标本50份.标准品采用WHO HBV DNA标准品.通过纳米磁珠实现对HBV核酸的吸附浓缩,提高提取的HBV核酸模板的浓度.以瑞士罗氏公司HBV DNA检测试剂和4种国产试剂原有的核酸提取检测方法为对照,评价该方法对国产核酸检测试剂的改进效果.结果纳米磁珠核酸提取方法与国产试剂结合后,国产试剂的分析灵敏度分别达到10和50 IU/ml,与罗氏试剂的分析灵敏度12 IU/ml基本相当.4种国产试剂盒内自带提取试剂检测临床乙型肝炎患者血清中痕量HBV DNA阳性检出率分别为64%(32份)、56%(28份)、62%(31份)和58%(29份),与罗氏试剂阳性检出率88%比较,差异具有统计学意义(x2值分别为7.895、12.698、9.013、11.416,P均＜0.05).纳米磁珠核酸提取方法与国产试剂结合后,阳性检出率分别为88%(44份)、88%(44份)、88%(44份)和86%(43份),与罗氏试剂(88%)比较,阳性检出率差异无统计学意义(x2值分别为0.000、0.000、0.000、0.088,P均＞0.05).HBV核酸浓度为101～103 IU/ml时,病毒核酸浓度对数值与循环阈值呈负相关,但是比103～106 IU/ml浓度范围的相关性下降.结论以纳米磁珠为介质建立的核酸提取方法可显著提高国产HBV DNA检测的分析灵敏度,对监测乙型肝炎患者血液中痕量HBV DNA含量具有重要意义.
건립일충기우납미자주위개질적핵산제취화부집방법,제고국산HBV핵산검측시제적분석령민도,용우흔량HBV DNA적측정.방법선택경항병독치료HBV DNA농도≤1×104 IU/ml적을형간염환자혈청표본50빈.표준품채용WHO HBV DNA표준품.통과납미자주실현대HBV핵산적흡부농축,제고제취적HBV핵산모판적농도.이서사라씨공사HBV DNA검측시제화4충국산시제원유적핵산제취검측방법위대조,평개해방법대국산핵산검측시제적개진효과.결과납미자주핵산제취방법여국산시제결합후,국산시제적분석령민도분별체도10화50 IU/ml,여라씨시제적분석령민도12 IU/ml기본상당.4충국산시제합내자대제취시제검측림상을형간염환자혈청중흔량HBV DNA양성검출솔분별위64%(32빈)、56%(28빈)、62%(31빈)화58%(29빈),여라씨시제양성검출솔88%비교,차이구유통계학의의(x2치분별위7.895、12.698、9.013、11.416,P균＜0.05).납미자주핵산제취방법여국산시제결합후,양성검출솔분별위88%(44빈)、88%(44빈)、88%(44빈)화86%(43빈),여라씨시제(88%)비교,양성검출솔차이무통계학의의(x2치분별위0.000、0.000、0.000、0.088,P균＞0.05).HBV핵산농도위101～103 IU/ml시,병독핵산농도대수치여순배역치정부상관,단시비103～106 IU/ml농도범위적상관성하강.결론이납미자주위개질건립적핵산제취방법가현저제고국산HBV DNA검측적분석령민도,대감측을형간염환자혈액중흔량HBV DNA함량구유중요의의.
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.