title: Minor groove binder modification of widely used TaqMan probe for hepatitis E virus reduces risk of false negative real-time PCR results.
abstract: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65-71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C→T single nucleotide mutation in the probe binding region in each case.
authors.affiliation: Division of Infection and Immunity, University College London, London, UK. j.garson@ucl.ac.uk
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Garson
authors.forename: Ruth B
authors.initials: RB
authors.lastname: Ferns
authors.forename: Paul R
authors.initials: PR
authors.lastname: Grant
authors.forename: Samreen
authors.initials: S
authors.lastname: Ijaz
authors.forename: Eleni
authors.initials: E
authors.lastname: Nastouli
authors.forename: Renata
authors.initials: R
authors.lastname: Szypulska
authors.forename: Richard S
authors.initials: RS
authors.lastname: Tedder
mesh_terms: D005260:Female; D016751:Hepatitis E / Q000175:diagnosis* / Q000821:virology; D016752:Hepatitis E virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D015345:Oligonucleotide Probes / Q000235:genetics*; D010949:Plasma / Q000821:virology; D011247:Pregnancy; D012367:RNA, Viral / Q000097:blood; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes; D012367:RNA, Viral