HCV-RNA detection in liver bioptates--a comparison of automatic and 'home-made' protocols combined with a new procedure of HCV-RNA extraction.
BACKGROUND
For HCV-RNA detection in liver tissue a generally accepted reference method has not been established yet. Therefore, we have developed a procedure of HCV-RNA extraction from liver tissue and compared two methods of HCV-RNA detection.


METHODS
A set of 32 liver biopsy specimens, obtained from chronically HCV infected patients, has been examined. At the time of biopsy, serum HCV-RNA was detectable in 20 patients in RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 (Roche Molecular Systems, Inc, Pleasanton, CA, USA) 2 serum samples were negative for HCV-RNA and 10 patients has not been tested. Liver tissue has been homogenized in the presence of CRSR-Green (Fast RNA Kit-Green, Bio101, Inc, Vista, CA, USA) and a mixture of phenol/chloroform/isoamylic alcohol, using a FastPrep homogenizator (Bio101, Inc, Vista, CA, USA). Then RNA has been isolated from the material obtained using the Total RNA Prep Plus procedure (A&A Biotechnology, Gdańsk, Poland). Presence of HCV-RNA was next tested by means either of 'home-made' nested RT-PCR procedure or the RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0. In case of an inhibition of PCR detected in the first run of automatic assay, both PCR procedures have been repeated.


RESULTS
In 5 cases of automatic assay an inhibition of PCR reaction has been detected in the first run, the RT-PCR procedures has been then successfully repeated in the second run. The presence of HCV-RNA in the liver tissues was detected in a total of 22 cases (69%) by mean of automatic assay and in a total of 20 cases (63%) by means of 'home-made' RT-PCR procedure. Except for two cases of HCV-RNA positivity in a biopsy tissue, detected by means of automatic but not 'home-made' procedure, the results obtained employing the methods have been identical. In one serum HCV-RNA positive case, the presence of HCV RNA has not been detected in a liver tissue using both methods.


CONCLUSIONS
Our procedure of RNA isolation combined either with automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 or 'home-made' RT-PCR procedure may be useful for establishing presence of HCV-RNA in liver tissue. In this combination, the automatic assay seems to be more sensitive than the 'home-made' procedure.
Bielawski
K P
KP
Molecular Diagnostics Division, Department of Biotechnology, Intercollegiate Faculty of Bioteçhnology, University of Gdańsk, Medical University of Gdańsk, ul. Kładki 24, 80-822 Gdańsk, Poland.
Bernat
A
A
Własiuk
M
M
Falkiewicz
B
B
D001706:Biopsy; D005821:Genetic Techniques; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D012367:RNA, Viral; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012367:RNA, Viral