Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler.
BACKGROUND
PCR techniques have proved to be more sensitive than traditional cell culture in the diagnosis of enterovirus and rhinovirus infections and are widely used in clinical virus laboratories. However, PCR assays are relatively time-consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings.


OBJECTIVE
The aim of the present study was to develop fast and sensitive real-time PCR assay, which would allow simultaneous detection of entero- and rhinoviruses and their quantification in clinical and experimental samples.


METHODS
Two real-time RT-PCR protocols were developed using LightCycler (LC) technology; SYBRGreen and hybridization probe assays. The sensitivity of these assays to detect entero- and rhinoviruses was compared with that of a traditional reference RT-PCR-hybridization assay and cell culture. All PCR protocols used the same primers amplifying the 5'-non coding region (NCR) of entero- and rhinoviruses. The LC probe assay and the reference RT-PCR used almost identical detection probes, which bind to enterovirus specific amplicons.


CONCLUSIONS
Both real-time PCR assays were equally sensitive as the reference RT-PCR-assay and all were more sensitive than cell culture. Both real-time assays quantified reliably the amount of the virus and took much shorter time than the reference RT-PCR. As the real-time SYBRGreen assay detects both entero- and rhinoviruses it can be used for primary screening of samples, which can be positive for either of these viruses. The real-time probe-assay can confirm the presence of enterovirus in SYBRGreen positive samples or it can be used for selective screening of enteroviruses e.g. from CSF samples.
Kares
Saara
S
Center of Laboratory Medicine, Tampere University Hospital, Teiskontie 35 PL 2000, 33521 Tampere, Finland. saara.kares@pshp.fi
Lönnrot
Maria
M
Vuorinen
Pauli
P
Oikarinen
Sami
S
Taurianen
Sisko
S
Hyöty
Heikki
H
D020121:5' Untranslated Regions / Q000032:analysis / Q000235:genetics; D052160:Benzothiazoles; D002555:Cerebrospinal Fluid / Q000821:virology; D015342:DNA Probes; D003959:Diamines; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology*; D005243:Feces / Q000821:virology; D006801:Humans; D009305:Nasopharynx / Q000821:virology; D009930:Organic Chemicals / Q000378:metabolism; D010850:Picornaviridae Infections / Q000175:diagnosis* / Q000821:virology*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012229:Rhinovirus / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019562:Viral Load
D020121:5' Untranslated Regions; D052160:Benzothiazoles; D015342:DNA Probes; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I