GENOTYPIC IDENTIFICATION OF RICKETTSIA-TSUTSUGAMUSHI BY RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS OF DNA AMPLIFIED BY THE POLYMERASE CHAIN-REACTION

We combined the nested polymerase chain reaction and restriction fragm ent length polymorphism (PCR-RFLP) for genotypic identification of Ric kettsia tsutsugamushi. Four primers were selected from the DNA sequenc e of the gene encoding a 56-kD serotype-specific antigen of the Karp s train. Nested PCR produced rickettsia-specific products of approximate ly 0.6 kb in the amplification of DNA prepared from three reference st rains (Gilliam, Karp, and Kato) and two prototype strains (Irie and Hi rano) prevalent in the Miyazaki Prefecture of Japan. When the nested P CR products obtained from these five strains were digested with Hha I, profiles specific to each strain were generated. Fourteen of 17 DNA s amples of peripheral blood mononuclear cells from patients with scrub typhus tested positive in the nested-PCR, providing a rickettsia-speci fic band. The serotype of infected rickettsia of 10 patients were diag nosed as Irie and those of four patients were diagnosed as Hirano by i ndirect immunofluorescence methods The fragment profiles of the PCR pr oducts of these 14 patients after digestion with Hha I corresponded cl osely with those serotypes. However, the PCR products from two of four samples, which were similar to Hirano strain by a serologic method an d by the pattern of digestion with Hha I, produced different RFLP prof iles upon further digestion with Hinf I and Alu I. These results may s uggest that genetic variation exists within serotypes. Genotypic ident ification of R. tsutsugamushi by means of PCR-RFLP using three restric tion enzymes is apparently useful.