Performance evaluation of HCMV DNA detection by a domestic magnetic bead nucleic acid automatic extraction system
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摘要: 目的:评价一种国产核酸磁珠自动提取系统对人巨细胞病毒(HCMV)DNA检测的性能。方法:采用科华磁珠核酸自动提取仪提取血浆HCMV DNA,经实时荧光定量PCR检测,分析其线性范围、灵敏度、精密度及抗干扰能力、8混养模式的检测效能。并利用30份含HCMV感染患者及20份健康人群血浆对该方法进行临床应用评价,其检测结果与沉淀离心提取法进行比较。结果:该方法检测HCMV DNA载量的线性范围为102~106 copy/mL。检测灵敏度为100 copy/mL(单检)。测定值与理论值的相关性良好,R2=0.998,P＜0.01。批内和批间变异系数(CV)均小于5%。临床应用评价发现,国产磁珠核酸自动提取法与沉淀离心提取法对临床样品的检测一致性较好(Kappa=0.839≥0.75,P＜0.01),磁珠自动提取法的阳性率高于沉淀离心提取法(100% vs 86.7%),但差异无统计学意义(P=0.125)。在102~103 copy/mL范围内,国产磁珠核酸自动提取发现优于沉淀离心法。2法所获得的定量结果差异有统计学意义(P＜0.05)。8混养模式的检测灵敏度为103 copy/mL,拆分结果与实际相符,未产生交叉污染。结论:基于国产磁珠自动核酸提取系统实现了快速、自动化提取HCMV DNA,具有良好检测性能,适用于采供血机构HCMV的血液核酸检测及临床应用推广。Abstract: Objective: To evaluate the performance of human cytomegalovirus (HCMV) DNA detection by a domestic magnetic bead extraction system.Method: The HCMV DNA was extracted from plasma by Kehua magnetic bead nucleic acid automatic extraction system and detected by real-time fluorescence quantitative PCR.The clinical application of this system was further evaluated with 30 plasma samples from HCMV infected patients and 20 plasma samples from healthy people.Result: The linear range of HCMV DNA concentration detection by this system was 102 to 106 copy/mL.The detection sensitivity was 100 copy/mL (individual test). The correlation between the measured value and theoretical value was perfect,R2=0.998,P＜0.01.The coefficient of variations (CV) within and between batches were all less than 5%.Clinical application evaluation found that the two nucleic acids extraction methods had good consistency (Kappa=0.839≥0.75,P＜0.01). Although the positive rate of magnetic beads automatic extraction group was higher than tha of the precipitation and centrifuge extraction group (100%vs 86.7%), which wasn't statistically significant difference (P=0.125). It was should noted that within the scope of 102 to 103 copy/mL,the nucleic acids extraction method by domestic magnetic beads automatic nucleic acids system was found better than precipitation and centrifuge extraction method.The quantitative results of the two methods was different (P＜0.05). The detection sensitivity of the 8 pool mode was 103 copy/mL,the resolution results were consistent with the actual results,and there was no cross contamination.Conclusion: Basing on the domestic magnetic bead automatic nucleic acid extraction system,the rapid and automatic extraction of HCMV DNA could be achieved,with good detection performance.It is not only suitable for the nucleic acid test of HCMV in blood centers,but also for the laboratory diagnosis of HCMV in hospitals.
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