Development of a high-throughput multiplexed real time RT-PCR assay for detection of human pegivirus 1 and 2
Development of a high-throughput multiplexed real time RT-PCR assay for detection of human pegivirus 1 and 2
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DOI：
10.1016/j.jviromet.2016.12.013
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发表时间：
2017-03-01
影响因子：
3.1
通讯作者：
Dawson, George J.
中科院分区：
文献类型：
--
作者：
Frankel, Matthew;Forberg, Kenn;Dawson, George J.
Human Pegivirus 2 (HPgV-2) was recently identified in the bloodstream of HCV-infected and multiply transfused individuals. Initial reports show HPgV-2 circulates at a low prevalence in HCV co-infected individuals, necessitating testing of large cohorts of samples to identify infected persons. The identification of additional HPgV-2 cases was facilitated by the development of a high throughput and reliable molecular reverse transcription polymerase chain reaction (RT-PCR) assay intended for use on the automated Abbott m2000 system with a capability of extracting and testing 96 samples at once. A dual target approach was taken to reduce the risk of a false-negative result, amplifying sequences within the 5' UTR and NS2/3 coding regions of HPgV-2. The assay was expanded to multiplex detection of the other human Pegivirus, HPgV-1 (formerly GBV-C), to allow simultaneous prevalence comparison. The limit of detection (LOD; 95% detection) for HPgV-2 was experimentally determined to be 126 copies/mL. Through use of the newly developed multiplex assay, 21 strains of HPgV-2 circulating in HCV past or present infections were identified, with all strains confirmed by next generation sequencing. The multiplexed assay has high specificity and showed no cross-reactivity of HPgV-2 with HPgV-1 or other Flaviviruses. This automated assay will be instrumental in future studies addressing HPgV-2 pathogenicity, prevalence, and sequence diversity. (C) 2016 Elsevier B.V. All rights reserved.
