The amounts of serum HBV DNA and HCV RNA were 39 copies/ml and 5 �� 107 copies/ml, respectively (patient 21) (34). In the noncancerous tissue from the patient with HBV and HCV coinfection, there was an intense hybridization signal for HCV RNA on RT-PCR-ISH in almost all the selleck compound hepatocytes (Fig. (Fig.5A,5A, panel b). There was also a positive but weak RT-PCR-ISH signal for HCV RNA in the tumor hepatocytes (Fig. (Fig.5B,5B, panel b). Few hepatocytes in the cancerous tissue were positive for HBV DNA by PCR-ISH (Fig. (Fig.5B,5B, panel a), and no HBV DNA hybridization signal was detected in the noncancerous tissue (Fig. (Fig.5A,5A, panel a). FIG. 5. Panels a and b, HBV DNA (panels a) and HCV RNA (panels b) in noncancerous (non-Ca) (A) and cancerous (Ca) (B) liver tissue obtained from a patient coinfected with HBV and HCV were detected by PCR-ISH (55 cycles of PCR) and RT-PCR-ISH (45 cycles of PCR), .

.. DISCUSSION The standard assay for detecting replication of HBV and HCV in tissue is ISH, but results are often inconsistent and sometimes difficult to reproduce. The specificity of ISH is high but its sensitivity low, and it is difficult to detect low copy numbers of the HBV or HCV genome in tissue. PCR technology has been adapted to in situ amplification of viral genomes or their replicative intermediates in liver tissue sections, but sensitivity and specificity remain major challenges to the application of this approach (13, 18, 23, 25, 26, 30, 31). Here, we describe the use of a novel, highly specific and sensitive PCR-ISH method to determine the distribution and localization of HBV DNA, HBV RNA, and HCV RNA in both normal and cancerous liver tissues.

PCR-ISH is the most sensitive technology currently available for the detection of viral genomes, but a major potential limitation of this approach is the low specificity. We were able to improve the specificity of PCR-ISH by careful optimization of certain steps. PCR was performed using sets of antisense and sense primers that were complementary to the sequences located in the S and X regions of HBV and the 5��-UTR upstream of the core region of HCV. We added PCR templates to the PCR mixture and then added the PCR mixture to the HBV- or HCV-negative tissue sections. The slides were placed in the GeneAmp in situ PCR system 1000 unit, and PCR-ISH was performed as described in Materials and Methods.

Following these results, we selected the primer and probe set that did not stain the HBV- or HCV-negative tissue sections by PCR-ISH. Second, the type and concentration of protease and the treatment time were adjusted to optimize permeabilization of membranes and release of protein-nucleic acid cross-linking while avoiding overdigestion. Cilengitide Third, to improve the specificity for detecting viral genomes, we limited the number of PCR cycles and fixed the liver tissue sections in 4% paraformaldehyde immediately after PCR amplification.