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首页> 外文期刊>Journal of Clinical Microbiology >Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay
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Concurrent Genotyping and Quantitation of Cytomegalovirus gB Genotypes in Solid-Organ-Transplant Recipients by Use of a Real-Time PCR Assay

机译：并发基因分型和定量使用实时荧光定量PCR技术检测固体器官移植受者中巨细胞病毒gB基因型

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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 log10 copies/ml) and very good precision (coefficient of variation, ～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.
机译：我们已经开发了一种实时基因分型和定量PCR（RT-GQ-PCR）测定法，用于对巨细胞病毒（CMV）进行基因分型，并同时对实体器官移植（SOT）受者中的病毒载量进行定量。基于gB基因中的序列多态性设计特殊的小沟DNA结合探针，以增加gB1至gB4的基因分型特异性。为了验证，测试了28个通过限制性片段分析（RFA）确定的具有已知基因型的样品和121个具有未知基因型的样品。所有样品均来自患有CMV病毒血症的SOT患者。通过使用RT-GQ-PCR和RFA确定的已知基因型，实现了100％的基因分型一致性。与RFA相比，RT-GQ-PCR鉴定出更多的混合基因型CMV感染病例。没有观察到基因型之间的交叉反应。在121个未知基因型样本中检测到所有四种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病毒载量明显高于单一基因型感染患者（ P = 0.019）。 RT-GQ-PCR检测方法具有很高的灵敏度和特异性，动态范围宽（2.7至10.7 log 10 拷贝/ ml），并且具有非常好的精密度（变异系数，约1.78％） ）。凭借在单个反应中同时进行CMV gB基因分型和定量的突出特点，该新方法为监测SOT接受者的CMV感染提供了一种快速且经济高效的方法。

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