Abstract
Conventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at a reference laboratory from 1979 to 2007; these common serotypes were identified using one sense and three antisense primers and a set of 80 serotype-specific probes in VP1 (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). In this study, one HEV-specific primer pair, two probes in the 5' untranslated region (UTR), and a new set of 80 serotype-specific probes in VP1 were designed. First, we successfully applied the modified RT-PCR-RLB (using two HEV-specific probes and two sets of serotype-specific probes) to synchronously detect the 5' UTR and VP1 regions of 131/132 isolates previously studied (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). Then, this method was used to identify 73/92 nonserotypeable HEV isolates; 19 nonserotypeable isolates were hybridized only with HEV-specific probes. The VP1 region of 92 nonserotypeable HEV isolates was sequenced; 73 sequences corresponded with one or both RLB results and 19 (not belonging to the 20 most common genotypes) were identified only by sequencing. Two sets of serotype-specific probes can capture the majority of strains belonging to the 20 most common serotypes/genotypes simultaneously or complementarily. Synchronous detection of the 5' UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, especially nonserotypeable isolates.
Publication types
-
Evaluation Study
-
Research Support, Non-U.S. Gov't
MeSH terms
-
DNA Primers / genetics
-
Enterovirus / classification*
-
Enterovirus / genetics*
-
Enterovirus / isolation & purification
-
Enterovirus Infections / virology*
-
Genotype
-
Humans
-
Molecular Sequence Data
-
Oligonucleotide Probes / genetics
-
Polymorphism, Genetic*
-
RNA, Viral / genetics
-
Reverse Transcriptase Polymerase Chain Reaction / methods*
-
Sequence Analysis, DNA
-
Serotyping
-
Virology / methods*
Substances
-
DNA Primers
-
Oligonucleotide Probes
-
RNA, Viral
Associated data
-
GENBANK/FJ868337
-
GENBANK/FJ868338
-
GENBANK/FJ868339
-
GENBANK/FJ868340
-
GENBANK/FJ868341
-
GENBANK/FJ868342
-
GENBANK/FJ868343
-
GENBANK/FJ868344
-
GENBANK/FJ868345
-
GENBANK/FJ868346
-
GENBANK/FJ868347
-
GENBANK/FJ868348
-
GENBANK/FJ868349
-
GENBANK/FJ868350
-
GENBANK/FJ868351
-
GENBANK/FJ868352
-
GENBANK/FJ868353
-
GENBANK/FJ868354
-
GENBANK/FJ868355
-
GENBANK/FJ868356
-
GENBANK/FJ868357
-
GENBANK/FJ868358
-
GENBANK/FJ868359
-
GENBANK/FJ868360
-
GENBANK/FJ868361
-
GENBANK/FJ868362
-
GENBANK/FJ868363
-
GENBANK/FJ868364
-
GENBANK/FJ868365
-
GENBANK/FJ868366
-
GENBANK/FJ868367
-
GENBANK/FJ868368
-
GENBANK/FJ868369
-
GENBANK/FJ868370
-
GENBANK/FJ868371
-
GENBANK/GU142867
-
GENBANK/GU142868
-
GENBANK/GU142869
-
GENBANK/GU142870
-
GENBANK/GU142871
-
GENBANK/GU142872
-
GENBANK/GU142873
-
GENBANK/GU142874
-
GENBANK/GU142875
-
GENBANK/GU142876
-
GENBANK/GU142877
-
GENBANK/GU142878
-
GENBANK/GU142879
-
GENBANK/GU142880
-
GENBANK/GU142881
-
GENBANK/GU142882
-
GENBANK/GU142883
-
GENBANK/GU142884
-
GENBANK/GU142885
-
GENBANK/GU142886
-
GENBANK/GU142887
-
GENBANK/GU142888
-
GENBANK/GU142889
-
GENBANK/GU142890
-
GENBANK/GU142891
-
GENBANK/GU142892
-
GENBANK/GU142893
-
GENBANK/GU142894
-
GENBANK/GU142895
-
GENBANK/GU142896
-
GENBANK/GU142897
-
GENBANK/GU142898
-
GENBANK/GU142899
-
GENBANK/GU142900
-
GENBANK/GU142901
-
GENBANK/GU142902
-
GENBANK/GU142903
-
GENBANK/GU142904
-
GENBANK/GU142905
-
GENBANK/GU142906
-
GENBANK/GU142907
-
GENBANK/GU142908
-
GENBANK/GU142909
-
GENBANK/GU142910
-
GENBANK/GU142911