Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing
Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing
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DOI：
10.1128/jcm.01605-13
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发表时间：
2013-11-01
影响因子：
9.4
通讯作者：
Pinsky, Benjamin A.
中科院分区：
文献类型：
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作者：
Sahoo, Malaya K.;Lefterova, Martina I.;Pinsky, Benjamin A.
巨细胞病毒（CMV）的抗病毒治疗在实体器官和造血干细胞移植受者的临床管理中起着重要作用。然而，CMV抗病毒治疗可能会因与磷酸转移酶UL 97和DNA聚合酶UL 54突变相关的耐药性而复杂化。我们开发了一种基于扩增子的高通量测序策略，用于检测临床血浆标本中的CMV耐药突变，使用微流体PCR平台进行多重文库制备和台式下一代测序仪器。使用UL 97和UL 54基因的质粒克隆来证明测定的低总体经验误差率（0.189%），并开发用于鉴定真实的低丰度变体的统计算法。使用野生型和突变型质粒的混合物以及已知含有赋予耐药性的突变的临床CMV分离株和血浆样本检测了该检测试剂盒检测耐药性突变的能力。最后，使用每次运行最多24份标本的多重方法，对48份病毒载量范围（394至2，191，011拷贝/ml血浆）的临床血浆标本进行测序。这导致了7个耐药突变的鉴定，其中3个存在于
Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in