title: Development of a high-throughput multiplexed real time RT-PCR assay for detection of human pegivirus 1 and 2.
abstract: 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.
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA. Electronic address: matthew.frankel@abbott.com.
authors.forename: Matthew
authors.initials: M
authors.lastname: Frankel
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: Kenn
authors.initials: K
authors.lastname: Forberg
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Coller
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Berg
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: John
authors.initials: J
authors.lastname: Hackett
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: Gavin
authors.initials: G
authors.lastname: Cloherty
authors.affiliation: Abbott Laboratories, Abbott Park, IL, USA.
authors.forename: George J
authors.initials: GJ
authors.lastname: Dawson
mesh_terms: D020121:5' Untranslated Regions; D057205:Automation, Laboratory; D060085:Coinfection / Q000821:virology; D018178:Flaviviridae Infections / Q000821:virology; D029407:GB virus C / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins
keywords: Automation; HCV; HPgV-2; PCR; Pegivirus; RT-PCR; Viral discovery