Diagnostic Assay forRickettsia japonica

Nozomu Hanaoka,Yoshinao Azuma,Ai Takano,Minenosuke Matsutani,Toshio Kishimoto,Motohiko Ogawa,Hiroki Kawabata,Seigo Yamamoto,Shuji Ando,Hiromi Fujita,Ichiro Kurane,Haruo Watanabe,Mutsunori Shirai,Akiko Sakata

doi:10.3201/eid1512.090252

Abstract

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains.

Highlights

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project
For determination of the DNA sequence for the 216-bp ORF, the primer pair of JapoSP5′ (5′-ACAACATCAATATTATAATTAGTATCC-3′) and JapoSP3′ (5′-TTCACGTATGTCTATATATGCTGCAG CG-3′) was used to amplify a 564-bp section, including this ORF, because this unique DNA sequence was located as the inserted sequence of the homolog for R. conorii RC1338 (Figure, panel A)
The nucleotide sequence of this ORF was identical among 5 of the R. japonica strains: DT-1, YH, FLA-1, HH-8, and HH-9 (100%); the sequence was highly conserved with significant identity in R. heilongjiangensis (99.5%), except for the Rickettsia sp

Summary

Diagnostic Assay for Rickettsia japonica

Results of this project show specific DNA regions for R. japonica in the Rickettsia genome One of these regions includes a 216-bp open reading frame (ORF) We focused on this conserved region of the 216-bp ORF to develop a TaqMan minor groove binder (MGB) probe (Applied Biosystems) that could detect the pathogenic R. japonica group, including R. heilongjiangensis, with a high degree of specificity. It could not detect R. rickettsii, R. prowazekii, or other Rickettsia strains These results indicate that the combination of probes and primers in this study had high specificity for the pathogenic R. japonica group. Eighteen DNA templates were extracted from blood clots collected from 18 patients in the acute stages of illness (male:female ratio 1:1; average age 64.1 years [range [27–88] years]); average number of days after onset

Conventional PCR

Findings

Conclusions