Diagnostic Assay forRickettsia japonica

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

doi:10.3201/eid1512.090252

Cited by 22 publications

(21 citation statements)

References 14 publications

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“…Several methods for the rapid molecular diagnosis of rickettsiosis based on genomic sequence data have been reported (3,(6)(7)(8)(9). However, a method that can simultaneously detect the DNA of several Rickettsia spp.…”

Section: Discussionmentioning

confidence: 99%

“…The genomic DNA from all the Rickettsia spp. was extracted as described in our previous study (6). Tris-EDTA buffer containing 100 mg/ml salmon sperm was used as the DNA dilution buffer to eliminate the absorption of DNA fragment in the microtube for the evaluation of sensitivity.…”

Section: Identificationmentioning

confidence: 99%

“…Thus, the LAMP assay is a simpler method for molecular diagnosis than the qPCR assay. In our previous study, we have developed a Rickettsia japonica-specific detection assay (6). For the detection of Rickettsia spp.…”

Section: Introductionmentioning

confidence: 99%

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Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Rickettsia spp.

et al. 2017

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SUMMARY:We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 639 C. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

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Section: Discussionmentioning

confidence: 99%

Section: Identificationmentioning

confidence: 99%

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Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Rickettsia spp.

et al. 2017

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“…We extracted DNA from O. tsutsugamushi -infected L-929 cell as mentioned in the previous section and performed the real-time PCR according to the general procedure [23]. We also used an IF staining to monitor the growth of O. tsutsugamushi .…”

Section: Methodsmentioning

confidence: 99%

Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics

et al. 2013

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BackgroundMycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro.ResultsWe cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 μg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 μg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 μg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 μg/ml as shown previously.ConclusionOur results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

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“…R. japonica was first identified as a causative agent of Japanese spotted fever [2] and has been isolated from patients in South Korea, the Philippines, and Thailand only in the past decade [3]–[5]. For rapid and accurate diagnosis of rickettsial infections, a new diagnostic assay of R. japonica using a species specific DNA sequence was proposed on the basis of a comparative genome analysis of rickettsiae isolated from Asian countries [6].…”

Section: Introductionmentioning

confidence: 99%

Complete Genomic DNA Sequence of the East Asian Spotted Fever Disease Agent Rickettsia japonica

et al. 2013

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Rickettsia japonica is an obligate intracellular alphaproteobacteria that causes tick-borne Japanese spotted fever, which has spread throughout East Asia. We determined the complete genomic DNA sequence of R. japonica type strain YH (VR-1363), which consists of 1,283,087 base pairs (bp) and 971 protein-coding genes. Comparison of the genomic DNA sequence of R. japonica with other rickettsiae in the public databases showed that 2 regions (4,323 and 216 bp) were conserved in a very narrow range of Rickettsia species, and the shorter one was inserted in, and disrupted, a preexisting open reading frame (ORF). While it is unknown how the DNA sequences were acquired in R. japonica genomes, it may be a useful signature for the diagnosis of Rickettsia species. Instead of the species-specific inserted DNA sequences, rickettsial genomes contain Rickettsia-specific palindromic elements (RPEs), which are also capable of locating in preexisting ORFs. Precise alignments of protein and DNA sequences involving RPEs showed that when a gene contains an inserted DNA sequence, each rickettsial ortholog carried an inserted DNA sequence at the same locus. The sequence, ATGAC, was shown to be highly frequent and thus characteristic in certain RPEs (RPE-4, RPE-6, and RPE-7). This finding implies that RPE-4, RPE-6, and RPE-7 were derived from a common inserted DNA sequence.

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Diagnostic Assay forRickettsia japonica

Cited by 22 publications

References 14 publications

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Rickettsia</i> spp.

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Rickettsia</i> spp.

Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics

Complete Genomic DNA Sequence of the East Asian Spotted Fever Disease Agent Rickettsia japonica

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