Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

AIM:To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

两种HBV DNA荧光定量PCR检测试剂的检测能力精度比较

目的:了解Roche公司Cobas PCR试剂与国产HBV荧光定量PCR试剂对HBV DNA检测的差异性,以更好地指导抗病毒治疗。方法:采用相关性分析来评价Cobas检测和国产检测对不同HBV DNA载量标本的精确性。结果:将全部标本的检测结果进行相关性分析,国产试剂与Roche试剂具有较好的相关性(r=0.612,P=0.004)。将标本分别按照以下5个病毒载量分级:10^(3)~10^(4),10^(3)~10^(5),10^(3)~10^(6),10^(3)~10^(7),10^(3)~10^(8)(copy/ml),则国产试剂检测结果与Roche定量结果的相关性分别为:相关系数r=-0.129,0.107,0.13,0.304,0.295,检测P值分别为:0.287,0.378,0.283,0.011,0.013。70例中国产试剂检测结果低于检测下限的标本有43例,而Roche检测结果处于检测下限的有17人(39.5%),余下26例(60.5%)的病毒载量均能检测到(处于10^(2)~10^(6) copy/ml范围内):其中,处于10^(2)~10^(3)的有10例,处于10^(3)~10^(4)的有9例,处于10^(4)~10^(5)的有2例,处于10^(5)~10^(6)范围内的有5例。国产试剂检测结果≤1000 copy/ml而Roche检测结果>1000 copy/ml的标本有16例,占37.2%。结论:总体上国产试剂与Roche试剂具有较好的相关性。病毒载量在10^(7) copy/ml以上的两者检测精度相似。国产试剂对病毒载量低于≤10^(6) copy/ml范围内的与Roche检测相关性较差,尤其低于国产检测下限的标本更需要采用Roche检测。

血液HBV全自动核酸筛查方法的应用研究

目的:探讨在加样仪上实现血液样本汇集,在RocheMPLC上全自动提取样本HBVDNA ,在COBASAM PLICOR上进行扩增和检测的方法,为血液HBV全自动核酸筛查提供应用依据。方法:在酶联免疫吸附实验(ELISA)筛查血液基础上,应用STAR2 0 0 0加样仪自编程序进行全自动血样汇集( 2 4人份) ,在MPLC上全自动方法提了样本核酸,应用PCR方法在COBASAMPLICOR进行扩增和检测分析结果,从检出限量及抗干扰能力方面进行考评。结果:用国际标准核酸质控品考评及深圳应用,表明全自动汇集,全自动核酸提取及扩增和检测HBVDNA 95 %检出限量为3 8.9IU/ml,95 %CI为2 1～3 2 3 ,2 0mg/dl胆红素、Ig/dl血色素及中等程度的脂血对结果无影响。通过对16 5 12个样本共688个汇集池分析,HBVDNA阳性8例,阳性率为0.0 4 9%。

Detection of Hepatitis B Virus M204V Mutation Quantitatively via Real-time PCR

Background and Aims:Drug-resistant DNA mutations of the hepatitis B virus(HBV)affect treatment response in chronic hepatitis B patients.We have established a new,sensitive,specific,accurate and convenient real-time PCR method to detect HBV mutations quantitatively.Methods:Blood samples were collected from patients showing viral breakthrough,primary nonresponse,or poor response during treatment,and mutations were detected via direct sequencing to assess our method.A plasmid containing the M204V mutation was synthesized and standard curves plotted.Results:The determination coefficient for linear correlation between Ct and log plasmid copy numbers was 0.996,where Ct value was−3.723log(DNA concentration)+48.647.Coefficients of variation indicated good reproducibility.Correctness was within tolerable bias.Limit of detection was 103 copies/mL.Specificity,accuracy,positive predictive value and negative predictive value were 92.86%,100%,96.88%,100%and 94.74%,respectively.Conclusions:These results show that our method can be used to detect HBV M204V mutations with the advantages of sensitivity,specificity and efficiency,providing a new choice for monitoring drug resistance.

不同方法检测血清HBV DNA水平的比较

目的:研究不同方法检测血清HBVDNA水平的比较。方法:采用151份标本分别采用COBAS Amplicor、实时定量PCR(LightCycler及Mx3000p)方法进行平行检测。结果:①检测灵敏度:COBAS Amplicor方法灵敏度高于实时PCR方法(P=0.005、0.014),而Mx3000p灵敏度高于LightCycler方法(P=0.042)。②相关性:实时定量PCR(LightCycler及Mx3000p)与COBAS Amplicor方法所测HBVDNA结果均呈线性相关(r=0.842,P<0.01;r=0.946,P<0.01)。结论:实时定量PCR方法是较为经济且准确的HBVDNA检测方法,而Mx3000p在准确性和灵敏度上可能优于LightCycler方法。