A real-time Taqman method for hepatitis C virus genotyping.
BACKGROUND
There is the need for a rapid, inexpensive method for genotyping hepatitis C virus (HCV) to support clinical practice.


OBJECTIVE
To develop a real-time (Rotor-Gene 3000) Taqman assay for HCV genotyping in a single tube.


METHODS
Seven type-specific probes, two for genotypes 1-3 and one for genotype 4 were designed around genotype-specific motifs in the 5' non-coding (NC) region to create two panels of probes. The first panel included two probes for genotype 1 detection and a single probe each for genotypes 2 and 3. The second panel had two probes for confirmation of genotypes 2 and 3 and a first line probe for genotype 4 detection. A comparative analysis of the Taqman assay against our in-house sequence-based method using 154 consecutive clinical samples, from HCV carriers in Cambridge, and four samples from the Quality Control for Molecular Diagnostics (QCMD) System was undertaken.


RESULTS
158 samples were analysed by conventional sequencing: 49% (n=78) were genotype 1, 11% (n=18) genotype 2, 30% (n=47) genotype 3 and 6% (n=10) genotype 4. For two samples, the sequence data was heterogeneous and difficult to analyse, suggesting mixed infection and for three samples, the viral load was insufficient for sequencing. Concordant results were obtained with the novel Taqman assay for 77/78 (99%) of genotype 1 isolates (positive with both genotype 1 probes), 17/18 (94%) of genotype 2 isolates, 43/47 (91%) of genotype 3 isolates and 10/10 (100%) genotype 4 isolates. One isolate, untypeable with sequencing was genotyped with the Taqman assay.


CONCLUSIONS
The Taqman assay was sensitive, specific and reliable over a wide range of viral loads and could identify mixed infections. These results highlight the potential of the Taqman assay as a fast, accurate and convenient method for routine HCV genotyping.
Rolfe
Kathryn J
KJ
Health Protection Agency, East of England Laboratory, Addenbrookes Hospital, Cambridge CB2 2QW, UK. kathryn.rolfe@addenbrookes.nhs.uk
Alexander
Graeme J M
GJ
Wreghitt
Tim G
TG
Parmar
Surendra
S
Jalal
Hamid
H
Curran
Martin D
MD
D020121:5' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D020121:5' Untranslated Regions; D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral