Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR
Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR
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DOI：
10.1128/jcm.42.3.1142-1148.2004
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发表时间：
2004-03-01
影响因子：
9.4
通讯作者：
Corey, L
中科院分区：
文献类型：
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作者：
Boeckh, M;Huang, M;Corey, L
先前的研究表明，检测血浆中巨细胞病毒（CMV） DNA的敏感性低于检测血液中巨细胞病毒的抗原血症试验。在1983份血液样本中，将三种不同引物组（UL125单独，UL126单独和UL55/ ul123 -外显子4）的血浆PCR检测与pp65抗原血症检测和血培养进行比较。血浆PCR在血液标本中检测巨细胞病毒的频率高于抗原血症试验或培养，但在三种PCR试验中，双引物试验（UL55/ ul123 -外显子4）在敏感性、特异性和预测值方面比抗原血症表现最好：151个抗原阳性样品中检测到122个（敏感性为80.1%），PCR阳性-抗原血症阴性样品中有122个（特异性为93%）。结果不一致的样本病毒载量较低（中位数为每张载玻片0.5个细胞；每毫升1150个拷贝），通常来自接受抗病毒治疗的患者。29份抗原血症阳性PCR阴性样本中有15份可通过其他方法检出巨细胞病毒，而122份PCR阳性抗原血症阴性样本中有121份可通过其他方法检出巨细胞病毒（P < 0.001）。在每个受试者的基础上，25例患者中有21例（抗原血症阳性-PCR阴性）和所有57例（PCR阳性-抗原血症阴性）在随访期间的不同时间点均可确诊。在对42例CMV血清阳性干细胞移植受者进行的事件时间分析中，双引物检测的灵敏度更高，与抗原血症相比，检测时间更早，三名CMV疾病患者中有两名抗原血症阴性，在发病前通过血浆PCR检测到。测定间变异性较低，动态范围为bbb50 log（10）。自动化DNA提取结果重复性高，CMV定量准确（R = 0.879 P < 0.001），灵敏度提高，样品处理速度加快。因此，引物优化和改进的DNA提取技术导致了基于血浆的PCR检测，其检测血液标本中CW的灵敏度明显高于pp65抗原血症和血培养。
Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.879 P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CW in blood specimens.