Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA
Scrub typhus is caused by an obligated intracellular organism, <i>Orientia tsutsugamushi</i> (<i>Orientia</i>). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serologi...
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| Format: | Article |
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MDPI AG
2019-07-01
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| Series: | Tropical Medicine and Infectious Disease |
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| Online Access: | https://www.mdpi.com/2414-6366/4/3/113 |
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| author | Chien-Chung Chao Tatyana Belinskaya Zhiwen Zhang Le Jiang Wei-Mei Ching |
| author_facet | Chien-Chung Chao Tatyana Belinskaya Zhiwen Zhang Le Jiang Wei-Mei Ching |
| author_sort | Chien-Chung Chao |
| collection | DOAJ |
| description | Scrub typhus is caused by an obligated intracellular organism, <i>Orientia tsutsugamushi</i> (<i>Orientia</i>). 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 <i>Orientia</i> 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 <i>Orientia</i> strains. The limit of detection is below single <i>Orientia</i> 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 <i>Orientia</i> infection. |
| first_indexed | 2024-04-11T20:43:44Z |
| format | Article |
| id | doaj.art-a8ed9e62bcae4324a9ee1e6f4c6e80a3 |
| institution | Directory Open Access Journal |
| issn | 2414-6366 |
| language | English |
| last_indexed | 2024-04-11T20:43:44Z |
| publishDate | 2019-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Tropical Medicine and Infectious Disease |
| spelling | doaj.art-a8ed9e62bcae4324a9ee1e6f4c6e80a32022-12-22T04:04:07ZengMDPI AGTropical Medicine and Infectious Disease2414-63662019-07-014311310.3390/tropicalmed4030113tropicalmed4030113Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNAChien-Chung Chao0Tatyana Belinskaya1Zhiwen Zhang2Le Jiang3Wei-Mei Ching4Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USADepartment of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USADepartment of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USADepartment of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USAViral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD 20910, USAScrub typhus is caused by an obligated intracellular organism, <i>Orientia tsutsugamushi</i> (<i>Orientia</i>). 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 <i>Orientia</i> 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 <i>Orientia</i> strains. The limit of detection is below single <i>Orientia</i> 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 <i>Orientia</i> infection.https://www.mdpi.com/2414-6366/4/3/113scrub typhus<i>Orientia tsutsugamushi</i>qPCRtraD genemultiple copy genediagnosis |
| spellingShingle | Chien-Chung Chao Tatyana Belinskaya Zhiwen Zhang Le Jiang Wei-Mei Ching Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA Tropical Medicine and Infectious Disease scrub typhus <i>Orientia tsutsugamushi</i> qPCR traD gene multiple copy gene diagnosis |
| title | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA |
| title_full | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA |
| title_fullStr | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA |
| title_full_unstemmed | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA |
| title_short | Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect <i>Orientia tsutsugamushi</i> DNA |
| title_sort | assessment of a sensitive qpcr assay targeting a multiple copy gene to detect i orientia tsutsugamushi i dna |
| topic | scrub typhus <i>Orientia tsutsugamushi</i> qPCR traD gene multiple copy gene diagnosis |
| url | https://www.mdpi.com/2414-6366/4/3/113 |
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