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Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA
Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies o...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789807/ https://www.ncbi.nlm.nih.gov/pubmed/31370347 http://dx.doi.org/10.3390/tropicalmed4030113 |
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author | Chao, Chien-Chung Belinskaya, Tatyana Zhang, Zhiwen Jiang, Le Ching, Wei-Mei |
author_facet | Chao, Chien-Chung Belinskaya, Tatyana Zhang, Zhiwen Jiang, Le Ching, Wei-Mei |
author_sort | Chao, Chien-Chung |
collection | PubMed |
description | Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating Orientia in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 Orientia strains. The limit of detection is below single Orientia genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of Orientia infection. |
format | Online Article Text |
id | pubmed-6789807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-67898072019-10-16 Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA Chao, Chien-Chung Belinskaya, Tatyana Zhang, Zhiwen Jiang, Le Ching, Wei-Mei Trop Med Infect Dis Article Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating Orientia in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 Orientia strains. The limit of detection is below single Orientia genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of Orientia infection. MDPI 2019-07-31 /pmc/articles/PMC6789807/ /pubmed/31370347 http://dx.doi.org/10.3390/tropicalmed4030113 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Chao, Chien-Chung Belinskaya, Tatyana Zhang, Zhiwen Jiang, Le Ching, Wei-Mei Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title_full | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title_fullStr | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title_full_unstemmed | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title_short | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA |
title_sort | assessment of a sensitive qpcr assay targeting a multiple-copy gene to detect orientia tsutsugamushi dna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789807/ https://www.ncbi.nlm.nih.gov/pubmed/31370347 http://dx.doi.org/10.3390/tropicalmed4030113 |
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