乙型肝炎病毒核酸的实时荧光定量PCR检测及其临床意义  

Clinical Significance of Detection of HBV-DNA Quantity by Real-time Fluorescence Quantitative PCR

作　　者：王旭东[1] 张冬雷[2] 李立人[3] 施健[2] 崔之础[2] 王惠民[1] 

机构地区：[1]南通大学附属医院检验医学中心,江苏南通226001 [2]南通大学附属医院酶学研究室,江苏南通226001 [3]南通大学附属医院消化内科,江苏南通226001

出　　处：《现代检验医学杂志》2006年第3期15-19,共5页Journal of Modern Laboratory Medicine

摘　　要：目的建立实时荧光定量PCR(rea l-tim e fluorescence quan titative PCR,RFQ-PCR)检测HBV-DNA含量的方法,探讨其在乙型肝炎预防、诊断、治疗及判断病情等方面的临床应用价值。方法在HBV S基因高保守区设计了特异性的引物和探针,利用T aqM an探针的基本原理,PCR扩增目的片段并实时检测产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中HBV-DNA的准确含量。结果220例临床样本的检测结果显示,乙肝大三阳患者HBV-DNA阳性率(98.8%)显著高于其它各组(P<0.01),病毒平均含量为7.52±0.43 cop ies/m l;小三阳患者HBV-DNA阳性率低于大三阳组(P<0.01),但平均病毒含量与大三阳组无差异(P>0.05),高达7.41±0.26 cop ies/m l;单独HB sA g阳性和单独HB sA b阳性组HBV-DNA阳性率分别为56.4%,19.2%,平均病毒含量分别为5.35±0.32 cop ies/m l,4.02±0.31 cop ies/m l;80例献血员血清仍有3例HBV-DNA阳性,其病毒含量低于其它各组。结论RFQ-PCR检测HBV-DNA含量比EL ISA法具有更好的灵敏度和特异性,能准确反映HBV在体内的复制情况,对临床上抗病毒药物的选择和判断疾病的预后意义重大。Objective To develop a new method for detection of HBV-DNA quantity by real-time fluorescence quantitative PCR （RFQ-PCR）,and to approach its clinical value in diagnosis,treatment,and prevention of HBV infection. Methods Specific primers and TaqMan probe had been designed in the highly conserved region of HBV S gene,and fluorescence of the PCR product was detected continuously during amplification. According to the standard curve created by plasmid DNA,the load of HBV DNA in clinical samples were determined using software. Results Serum samples from 220 hepatitis patients were tested by RFQ-PCR and ELISA simultaneously. The results showed that in the first group with HBsAg＋, HBeAg ＋and HBcAb＋ samples, the positive rate of HBV-DNA （98.8% ） was significantly higher than any other groups （P〈0.01） ,and the mean load of HBV-DNA was 7.52±0. 43 copies/ml; in the second group with HBsAg＋ ,HBeAb＋and HBcAb＋samples,the positive rate of HBV-DNA was lower （P〈0. 01）. But there was no significant difference of the mean load of HBV-DNA between the first and the second group（P〉0. 05）. In the group with HBsAg＋and the group with HBsAb＋ ,the positive rates were 56.44%and 19.2%, the mean loads were 5. 35±0.32 copies/ml and 4. 02±0. 31 copies/ ml. respectively. In 80 blood donors ,there were still 3 cases with HBV-DNA positive,but the mean load of HBV-DNA was much lower than any other groups. Conclusion The results showed RFQ-PCR was more sensitive and specific than ELISA,and it can be used as a good monitor for the state of HBV infection and its complication.

关 键 词：乙型肝炎病毒 实时荧光定量PCR 定量 

分 类 号：Q503[生物学—生物化学] R512.62[医药卫生—内科学]