Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of &lt;i&gt;Rickettsia&lt;/i&gt; spp.

Hanaoka, Nozomu; Matsutani, Minenosuke; Satoh, Masaaki; Ogawa, Motohiko; Shirai, Mutsunori; Ando, Shuji

doi:10.7883/yoken.jjid.2015.579

Cited by 5 publications

(3 citation statements)

References 14 publications

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“…Currently, the application of LAMP for the molecular detection of Rickettsia spp., scrub typhus and murine typhus, has been reported (Dittrich et al, 2014;Hanaoka et al, 2017;Roy et al, 2021). According to the criteria of the World Health Organization (WHO, 2021), the new LAMP technique for R. rickettsii meets many of the requirements of a molecular detection method, including low cost, simplicity, speed, robustness, and easy availability of instruments and equipment.…”

Section: Discussionmentioning

confidence: 99%

Development and validation of a novel detection method for Rickettsia rickettsii using a loop-mediated isothermal amplification assay

Carvajal-Gamez,

Olguín-Barrera,

Tinoco-Gracia

et al. 2024

Front. Microbiol.

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IntroductionRickettsia rickettsii is an obligate, intracellular pathogen and the causative agent of Rocky Mountain spotted fever (RMSF). RMSF is an important zoonotic disease due to its high fatal outcome in humans. The difficulty of clinical diagnosis due to the low sensitivity and specificity of current diagnostic methods are a principal setback. We reported the development of a new method for the detection of R. rickettsii in human and tick DNA samples using loop-mediated isothermal amplification (LAMP), as well as the validation of the LAMP test for R. rickettsii in field samples of infected ticks and humans, determining the diagnostic sensitivity and specificity, as well as the reproducibility of the test.MethodsThis technique uses hydroxy naphthol blue (HNB) as an indicator of the formation of magnesium pyrophosphate, a marker for the presence of DNA. Here, we used a putative R. rickettsii gene as a target for three pairs of primers that specifically amplify R. rickettsii DNA by hairpin-based isothermal amplification technique (LAMP).Results and discussionThe sensitivity of the assay was ~1.6–3 pg, which is 10 times more sensitive than PCR. To determine the diagnostics specificity and sensitivity, 103 human DNA samples and 30 tick DNA samples were evaluated. For the human samples, a sensitivity for HNB of 93%, a specificity of 70% and a k of 0.53 were obtained. For electrophoresis the sensitivity was 97% with a specificity of 58% and a k of 0.42. For tick samples, a sensitivity of 80% was obtained, a specificity of 93% for HNB and for electrophoresis the sensitivity and specificity were 87%. The k for both was 0.73. The degree of concordance between HNB and electrophoresis was 0.82 for humans and for ticks, it was 0.87. The result is obtained in shorter time, compared to a PCR protocol, and is visually interpreted by the color change. Therefore, this method could be a reliable tool for the early diagnosis of rickettsiosis.

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Section: Discussionmentioning

confidence: 99%

Development and validation of a novel detection method for Rickettsia rickettsii using a loop-mediated isothermal amplification assay

Carvajal-Gamez,

Olguín-Barrera,

Tinoco-Gracia

et al. 2024

Front. Microbiol.

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“…Besides, Hanaoka et. al found that the limit of detection for rickettsiae was 100 copies/μL by LAMP method [ 39 ], while the detection limit of CDA assay was 10 copies/μL. Furthermore, the sensitivity and specificity were both 100% from total 416 tests by CDA assay, which were superior to LAMP ( Table 3 ).…”

Section: Discussionmentioning

confidence: 99%

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

Gong

Cai

et al. 2022

PLoS Negl Trop Dis

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Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii (R. raoultii), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R. raoultii is in urgently need. In this study, a R. raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A (OmpA) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R. raoultii. Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R. raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care.

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“…The loop-mediated isothermal amplification assay is such a method and offers the ability to detect fewer than 100 copes of DNA per reaction. 80,81 Recombinase polymerase amplification assays are also inexpensive with field applicability for use in resource-limited regions. 82 Unfortunately, these methods will likely suffer from the aforementioned clinical limitations of PCR when used on peripheral blood specimens.…”

Section: Diagnosismentioning

confidence: 99%

The Rickettsioses

Blanton

2019

Infectious Disease Clinics of North America

129

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Rickettsia are small, obligately intracellular Gram negative bacilli. They are distributed among a variety of hematophagous arthropod vectors and cause illness throughout the world. Rickettsioses present as an acute undifferentiated febrile illness and are often accompanied by headache, myalgias, and malaise. Cutaneous manifestations include rash and eschar, which both occur at varying incidence depending on the infecting species. Serology is the mainstay of diagnosis, and the indirect immunofluorescence assay is the test of choice. Reactive antibodies are seldom present during early illness, so testing should be performed on both acute-and convalescent-phase sera. Doxycycline is the treatment of choice.

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

Cited by 5 publications

References 14 publications

Development and validation of a novel detection method for Rickettsia rickettsii using a loop-mediated isothermal amplification assay

Development and validation of a novel detection method for Rickettsia rickettsii using a loop-mediated isothermal amplification assay

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii

The Rickettsioses

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