Performance of enterovirus genotyping targeting the VP1 and VP2 regions on non-typeable isolates and patient specimens

Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR...

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Veröffentlicht in:Journal of virological methods 2010-04, Vol.165 (1), p.46-50
Hauptverfasser: She, Rosemary C., Hymas, Weston C., Taggart, Edward W., Petti, Cathy A., Hillyard, David R.
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Hymas, Weston C.
Taggart, Edward W.
Petti, Cathy A.
Hillyard, David R.
description Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates ( n = 36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies ( n = 50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5′-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. Using a one-step RT-PCR without nesting, most enterovirus isolates and a subset of clinical samples with high viral titer could be genotyped.
doi_str_mv 10.1016/j.jviromet.2009.12.016
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Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates ( n = 36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies ( n = 50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5′-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. 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subjects Algorithms
Biological and medical sciences
Cerebrospinal Fluid - virology
Coxsackievirus
Echovirus
Enterovirus - classification
Enterovirus - genetics
Enterovirus - isolation & purification
Enterovirus 68
Enterovirus Infections - virology
Fundamental and applied biological sciences. Psychology
Humans
Microbiology
Plasma - virology
Poliovirus
Reverse Transcriptase Polymerase Chain Reaction - methods
Sensitivity and Specificity
Serotype
Techniques used in virology
Virology
title Performance of enterovirus genotyping targeting the VP1 and VP2 regions on non-typeable isolates and patient specimens
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