Self-assembly formed by a short DNA probe pair: Application for highly sensitive mRNA species detection without reverse transcription.

We describe a novel technology for detecting nucleic acids: Probe Alteration Link Self-Assembly Reactions (PALSAR). PALSAR comprises DNA self-assembly of pairs of short DNA probes formed by alternate hybridization of three complementary regions in a pair of honeycomb probes (HCPs). Self-assembly occurs at designated salt concentrations and reaction temperatures and requires no enzymes. We prepared pairs of HCPs to detect mRNAs encoded by the GAPDH gene ß-actin (BA) gene, CD3D gene, CD4 gene, major vault protein (MV) gene and the signalling lymphocytic activation molecule-associated protein (SAP) gene, and succeeded in quantitatively detecting these mRNAs. PALSAR could detect mRNA directly without synthesizing cDNA. Moreover, multiple mRNAs could be detected simultaneously in a single reaction tube and there was a good correlation between the results obtained PALSAR and those by real-time PCR.

Full-length sequence of hepatitis B virus belonging to genotype H identified in a Japanese patient with chronic hepatitis.

We have isolated and cloned the full-length nucleotide sequence of the hepatitis B virus (HBV) genome (denoted HBV-IM806-2) recovered from a Japanese patient with chronic hepatitis. This patient had a history of travel to Bangkok, Thailand, and then suffered the onset of acute hepatitis B 3 months after his return to Japan. The HBV-IM806-2 isolate was composed of 3,215 nucleotides and showed the highest similarity to genotype H of HBV. Interestingly, 24 amino acid residues specific for genotype H were identified throughout the full genome sequence. Furthermore, phylogenetic analysis based on the full genome sequence confirmed that IM806-2 belonged to genotype H and was more closely related to the prototype of the Los Angeles strain than to the Nicaragua strain.

Present state of hepatitis E virus epidemiology in Tokyo, Japan.

Recently studies have reported the possibility that an indigenous hepatitis E virus (HEV) exists in Japan, but the epidemiological features of HEV in Japan are inadequate to make a judgment. In order to search the present state of HEV infection in Japan, we used ELISA to test 1033 sera from residents living in Tokyo and the Tokyo suburbs, for the presence of the antibody against HEV. The positive rate of anti-HEV IgG was 15.4% in all liver disease patients (68 of 440), 3% (6/200) in healthy individuals and 0.4% in infants (1/246), respectively (P<0.01). Anti-HEV IgG was seen in 17.6% (35/199) of liver disease patients of unknown etiology; 29.4% (5/17) of fulminant hepatitis, 17% of acute hepatitis (15/88) and 16% of chronic hepatitis (15/94). Anti-HEV IgG co-existed with hepatitis B virus and hepatitis C virus in 23.6% (21/89) and 7.9% (12/152), respectively. Furthermore, the prevalence of anti-HEV IgG was significantly higher in hemodialysis patients (18/60: 30%) and hospital workers (8/87: 9.2%) than in the healthy population (P<0.01). Anti-HEV IgM was detected in 0.1% of all samples tested (1/1033). The prevalence of anti-HEV IgG increased with age. No individuals with HEV antibody had a recent history of visiting countries where hepatitis E is endemic. These results indicate that generally 15.4% of Japanese patients with liver diseases had a history of HEV infection in the past. The routes of transmission of HEV require clarification in Japan.