Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3).
Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prev...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2020-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0241420 |
| _version_ | 1818588007144357888 |
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| author | Finn N Loose André Breitbach Ivo Bertalan Dana Rüster Uwe Truyen Stephanie Speck |
| author_facet | Finn N Loose André Breitbach Ivo Bertalan Dana Rüster Uwe Truyen Stephanie Speck |
| author_sort | Finn N Loose |
| collection | DOAJ |
| description | Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity. |
| first_indexed | 2024-12-16T09:17:54Z |
| format | Article |
| id | doaj.art-ee695c99a92645339dabd7d004e8f93d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-16T09:17:54Z |
| publishDate | 2020-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-ee695c99a92645339dabd7d004e8f93d2022-12-21T22:36:52ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-011510e024142010.1371/journal.pone.0241420Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3).Finn N LooseAndré BreitbachIvo BertalanDana RüsterUwe TruyenStephanie SpeckKoi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.https://doi.org/10.1371/journal.pone.0241420 |
| spellingShingle | Finn N Loose André Breitbach Ivo Bertalan Dana Rüster Uwe Truyen Stephanie Speck Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). PLoS ONE |
| title | Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). |
| title_full | Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). |
| title_fullStr | Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). |
| title_full_unstemmed | Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). |
| title_short | Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3). |
| title_sort | diagnostic validation of a rapid and field applicable pcr lateral flow test system for point of care detection of cyprinid herpesvirus 3 cyhv 3 |
| url | https://doi.org/10.1371/journal.pone.0241420 |
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