Comparison of transcription mediated amplification (TMA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of hepatitis C virus RNA in liver tissue.
BACKGROUND
Transcription mediated amplification (TMA) is known to be one of the most sensitive detection assays for hepatitis C virus (HCV) RNA in serum but has not yet been evaluated in liver tissue. It is unknown whether the higher sensitivity of TMA in comparison with polymerase chain reaction (PCR)-based assays is related to a higher efficiency of the extraction and/or amplification step.


OBJECTIVE
The sensitivity of a TMA-based assay (Versant HCV RNA Qualitative assay, Bayer Diagnostics) and a standard RT-PCR-based assay (Cobas Amplicor HCV 2.0, Roche Diagnostics) was compared in formalin-fixed paraffin-embedded liver biopsy specimens of patients with chronic hepatitis C.


METHODS
After deparaffinization of 7.5 microm liver sections HCV RNA was extracted by standard phenol/chloroform. HCV RNA dilution panels were transferred in parallel to cDNA synthesis and amplification steps of PCR and TMA. Furthermore, TMA amplification from stepwise diluted HCV sera was performed following RNA extraction by either microcentrifuge colums (QIAmp Viral RNA spin Kit, Qiagen, Hilden, Germany) or magnetic microparticles (VERSANT HCV RNA Qualitative assay).


RESULTS
The total number of HCV RNA positive liver specimens detected by TMA was higher compared with those detected by RT-PCR (P=0.032). The total number of TMA positive serum samples was higher when HCV RNA was extracted using magnetic microparticles in comparison with multicentrifuge column extraction (P=0.019).


CONCLUSIONS
Our results suggest that both the extraction and amplification step of the TMA-based assay contribute to the higher sensitivity compared with standard RT-PCR.
Hofmann
Wolf Peter
WP
Klinik für Innere Medizin II, Universität des Saarlandes, 66421 Homburg, Germany.
Dries
Volker
V
Herrmann
Eva
E
Gärtner
Barbara
B
Zeuzem
Stefan
S
Sarrazin
Christoph
C
D005557:Formaldehyde; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D019698:Hepatitis C, Chronic / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016612:Paraffin Embedding; D012367:RNA, Viral / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D005557:Formaldehyde