Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA.
BACKGROUND
Tick borne encephalitis virus (TBEV), is a human flavivirus causing tick borne encephalitis (TBE), a viral infection of the central nervous system endemic in Europe and Asia.


OBJECTIVE
To develop a reverse transcription polymerase chain reaction (RT-PCR) assay based on quantitative real-time RT-PCR technology (TaqMan) for detection and quantification of TBEV RNA. The test includes an internal control (IC) to avoid false negative results.


METHODS
The system was established and validated using wild-type (WT) non-infectious synthetic RNA representing a fragment of the 3' non-coding region of the TBEV genome. In addition, synthetic RNA differing from the WT synthetic RNA by a unique probe binding region was used as IC to monitor the overall efficiency of the RT-PCR.


RESULTS
The analytical sensitivity of the assay was at least ten copies of the TBEV synthetic transcript in presence of 50 copies of the IC. Successful amplification was obtained for different strains within the TBEV complex (Hypr, Hochosterwitz, Laibach, Elsass=Alsace, ZZ9, Wladiwostok). Among 14 serum and 21 cerebrospinal fluid (CSF) samples obtained from 28 patients with clinical suspicion of TBEV 1 CSF sample tested positive for TBEV RNA. In addition, no TBEV RNA could be detected in blood samples obtained from three vaccinated people 1 and 3 days post-vaccination. Thus indicating that a positive result is unlikely to be caused by recent vaccination.


CONCLUSIONS
A quantitative, highly sensitive and specific real-time RT-PCR assay has been developed for the detection of TBEV RNA. Inclusion of an IC is important to monitor the possible occurrence of false-negative results caused by the presence of inhibitory factors. This assay should be an important asset for the routine laboratory detection of TBEV RNA.
Schwaiger
Michaela
M
Institute for Clinical Microbiology and Immunology, Frohbergstr. 3, 9001, St. Gallen, Switzerland.
Cassinotti
Pascal
P
D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000302:isolation & purification; D004675:Encephalitis, Tick-Borne / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid / Q000592:standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D019914:Taq Polymerase / Q000378:metabolism
D020413:3' Untranslated Regions; D017931:DNA Primers; D012367:RNA, Viral; D019914:Taq Polymerase