Aim: The study aimed to clarify the advantages and disadvantages of different detection methods for JC virus (JCV) in human tissue specimens.
Methods: Specimens of lung and gastric carcinomas, normal lung tissue and gastric mucosa, and tonsil were examined for T antigen, VP and agnoprotein of JCV by nested-PCR, Southern blotting and sequencing. In addition, JCV load targeting T antigen was evaluated by real-time PCR and JCV existence morphologically by immunohischemistry, in situ hybridization (ISH) and PCR. For these experiments, the JCI cell line (JCV cultured neuroblastoma cell line) was employed as positive control.
Results: In lung and gastric carcinomas, T-antigen, VP and agnoprotein of JCV could be detected by nested PCR whose products were confirmed by Southern blots and sequencing. With real-time PCR, frozen samples of gastric carcinomas gave better detection of JCV than their corresponding paraffin-embedded tissues (P<0.05). Positive rate of JCV was high in lung carcinoma, compared with the normal lung tissue (P<0.05). It was the same for JCV copies in gastric carcinoma (P<0.05). Only the positive control exhibited JCV in the nucleus by ISH and T antigen in the cytoplasm by immunohistochemistry. In situ PCR demonstrated that JCV genomic DNA was located in the nucleus of the carcinoma cell, some alveolar epithelial cells, and tonsil lymphocytes. In in situ hybridization and PCR, NBT/BCIP coloring was stronger and Fuchsin.
Conclusions: Nested PCR whose amplicons should be confirmed by Southern blot and sequencing was a comparatively sensitive approach to detect JCV genomic DNA in human non-neural tissues. Real-time PCR might be employed to quantify copy number of JCV. In situ PCR was a good method to observe the JCV location in cells, given appropriate modulation of amplification cycles. Combination of various approaches will be adopted to explore the oncogenic roles of JCV in malignancies.
- JC virus
- tissue specimen