Pang Xiaoli, Humar Atul, Preiksaitis Jutta K
Provincial Laboratory for Public Health Microbiology, University of Alberta Hospital, Edmonton, AB, Canada.
J Clin Microbiol. 2008 Dec;46(12):4004-10. doi: 10.1128/JCM.01341-08. Epub 2008 Oct 29.
We have developed a real-time genotyping and quantitative PCR (RT-GQ-PCR) assay to genotype cytomegalovirus (CMV) and quantify viral loads simultaneously in solid organ transplant (SOT) recipients. Special minor-groove DNA-binding probes were designed based on sequence polymorphism in the gB gene to increase genotyping specificity for gB1 to gB4. For validation, 28 samples with known genotypes determined by restriction fragment analysis (RFA) and 121 with unknown genotypes were tested. All samples were from SOT patients with CMV viremia. A 100% concordance for genotyping was achieved by using the RT-GQ-PCR with known genotypes determined by RFA. The RT-GQ-PCR identified more cases of CMV infections with mixed genotypes than RFA did. No cross-reaction between genotypes was observed. All four gB genotypes were detected in the 121 samples of unknown genotype. gB1 was the predominant single genotype (n = 61, 50.4%), followed by gB2 (n = 26, 21.0%), gB3, (n = 11, 9.1%), and gB4 (n = 3, 2.5%). Mixed-genotype infections were detected in 17% (20/121) of the samples. Patients with mixed-genotype infections had significantly higher CMV viral loads than those with single-genotype infections (P = 0.019). The RT-GQ-PCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.7 to 10.7 log(10) copies/ml) and very good precision (coefficient of variation, approximately 1.78%). With the prominent feature of concurrent CMV gB genotyping and quantitation in a single reaction, the new assay provides a rapid and cost-effective method for monitoring CMV infection in SOT recipients.
我们开发了一种实时基因分型和定量聚合酶链反应（RT-GQ-PCR）检测方法，用于对实体器官移植（SOT）受者的巨细胞病毒（CMV）进行基因分型，并同时定量病毒载量。基于gB基因的序列多态性设计了特殊的小沟DNA结合探针，以提高对gB1至gB4的基因分型特异性。为进行验证，对28份通过限制性片段分析（RFA）确定了已知基因型的样本和121份基因型未知的样本进行了检测。所有样本均来自患有CMV病毒血症的SOT患者。通过使用RT-GQ-PCR与通过RFA确定的已知基因型进行基因分型，一致性达到了100%。与RFA相比，RT-GQ-PCR检测出更多混合基因型的CMV感染病例。未观察到基因型之间的交叉反应。在121份基因型未知的样本中检测到了所有四种gB基因型。gB1是主要的单一基因型（n = 61，50.4%），其次是gB2（n = 26，21.0%）、gB3（n = 11，9.1%）和gB4（n = 3，2.5%）。在17%（20/121）的样本中检测到混合基因型感染。混合基因型感染患者的CMV病毒载量显著高于单一基因型感染患者（P = 0.019）。发现RT-GQ-PCR检测方法具有高度敏感性和特异性，动态范围宽（2.7至10.7 log(10)拷贝/毫升）且精密度非常好（变异系数约为1.78%）。该新检测方法具有在单一反应中同时进行CMV gB基因分型和定量的突出特点，为监测SOT受者的CMV感染提供了一种快速且经济高效的方法。
