实时定量RT-PCR对乙型肝炎病毒全长RNA(fRNA)的定量检测

目的:建立一种简便的定量检测慢性乙型肝炎患者血清中终止于聚腺苷酸化位点的乙型肝炎病毒全长RNA(f RNA)的方法。方法:选取53例未治疗的乙型肝炎患者及22例HBs Ag阴性的健康者为研究对象,使用锚定oligo-d T的引物对其血清中f RNA进行实时定量反转录PCR检测,统计分析其与HBV DNA、HBcr Ag和HBe Ag的相关性。结果:对f RNA进行实时荧光定量RT-PCR检测的下线为2.3 log copies/ml,标准曲线的相关系数为0.99(P0.0001)。53例乙型肝炎患者中,29例(54.7%)可以检测到f RNA,22例正常对照中没有检测到f RNA。27例HBe Ag阳性和/或高水平HBV DNA的患者全部检测到f RNA,26例HBe Ag阴性并且低水平HBV DNA的乙型肝炎患者中有2例(7.7%)检测到f RNA(P0.0001)。HBe Ag阳性患者血清中f RNA水平高于HBe Ag阴性患者(5.0±0.3 vs.2.9±0.4 log copies/ml,P0.001)。f RNA与HBV DNA/HBcr Ag具有显著相关性(r=0.905、0.881,P0.0001)。Hayashi's定量分析法I显示f RNA与HBV DNA相关性强于其与HBcr Ag的相关性。结论:与HBV DNA和HBe Ag一样,f RNA可作为常规检测判断HBV的复制水平并指导用药。

Validation of a Simple Real-time RT-PCR for Detection and Quantization of HBV fRNA

Objective:To establish a simple assay for the detection and quantization of full-length RNA (fRNA) terminating at polyadenilation site in sera of chronic hepatitis B (CHB) patients.Methods:fRNA were assayed via TaqMan real-time RT/PCR using anchored oligo-dT primers in sera of 53 treatment-naive CHB patients and 22 HBsAg-negative healthy controls. Results were analyzed by comparation of HBV DNA with HBcrAg and HBeAg.Results:The fRNA assay had a lower limit of detection and quantization at 2.3 log copies/ml, and a correlation coefficient of 0.99 (P<0.0001). fRNA was detected in 29 of 53 (54.7%) of the CHB patients as compared to non of 22 controls (Specificity). fRNA was detected in all 27 HBeAg-positive and/or high HBV DNA levels CHB patients as compared to 2 of 26 (7.7%) HBeAg-negative and low HBV DNA levels CHB patients (P<0.0001). fRNA levels were higher in HBeAg-positive than in HBeAg-negative samples (5.0± 0.3 vs. 2.9± 0.4 log copies/ml, P<0.001). Significant correlation was found between fRNA and HBV DNA/ HBcrAg (r=0.905 and 0.881, respectively, P<0.0001). The effective items on fRNA levels, in descending order, were: HBV DNA, HBcrAg by means of Hayashi''s quantification method type I (Multiple correlation efficient=0.939).Conclusion:The simple real-time RT/PCR for detection and quantization of fRNA was suitable for routine clinical test in assessing HBV replication status the same as HBV DNA and HBeAg in CHB patients.