Detection and typing of TT virus DNA genotype by the PCR-RFLP method

TT virus (TTV) is a novel single-stranded DNA virus that was identified in patients with post-transfusion-hepatitis of non-A-G type in Japan in 1997. We developed a new detection and typing method for TTV DNA strains, by combining polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) techniques. TTV DNA was amplified by nested-PCR with TTV-specific mixed primers derived from the conserved open reading frame 1 (ORF1) region of the published TTV DNA sequences. Using the enzymes Hae III, Dra I, Eco RI and Pst I, we were able to distinguish between the four TTV genotypes. In 200 serum samples obtained from healthy pregnant women at an outpatient clinic, the positive rate of TTV detection was 27.0%, almost identical to the positive rate of the detection of ORF1-PCR in Japanese healthy individuals. The strains detected in the positive serum samples were genotype 1a (9.3%), genotype 1b (11.1%), genotype 2 (51.9%), genotype 3 (7.4%) and other genotypes (1.9%). This results indicate that genotype 1 and genotype 2 were the two major strains in the Japanese population. Moreover, 10 serum samples (18.5%) presented RFLP patterns in which some genotypes were mixed.