Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions
Background: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. Objectives: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 50 untranslated region (50 UTR)/exon 1 of the tat gene of EIAV. Study design: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RTPCR (RT-qPCR) along with the AGID test. Methods: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 50 UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. Results: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. Main limitations: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. Conclusions: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or “serologically silent” equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions.
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Wiley
2018
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Subjects: | Caballos, Virus de los Animales, Horses, Equine Infectious Anaemia, Diagnosis, Diagnóstico, Anemia Infecciosa Equina, PCR, Animal Viruses, Equinos, Insulated Isothermal RT-PCR, Point-of-Need Testing, |
Online Access: | http://hdl.handle.net/20.500.12123/4481 https://www.onlinelibrary.wiley.com/doi/10.1111/evj.13032 https://doi.org/10.1111/evj.13032 |
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oai:localhost:20.500.12123-44812019-06-06T18:07:36Z Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions Cook, R.F. Barrandeguy, Maria Edith Lee, Pei-Yu Alison Tsai, Chuan-Fu Shen, Yu-Han Tsai, Yun-Long Chang, Hsiao-Fen G. Wang, Hwa-Tang Thomas Balasuriya, Udeni B.R. Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing Background: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic. Objectives: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 50 untranslated region (50 UTR)/exon 1 of the tat gene of EIAV. Study design: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RTPCR (RT-qPCR) along with the AGID test. Methods: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 50 UTR/tat gene and samples from two EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR and AGID using samples derived from 196 inapparent EIAV carrier horses. Results: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and 50.00%, respectively, when compared to the AGID test. Main limitations: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests. Conclusions: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently exposed or “serologically silent” equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore, the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions. Instituto de Virología Fil: Cook, R.F. Louisiana State University. School of Veterinary Medicine. Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences; Estados Unidos Fil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; Argentina Fil: Lee, Pei-Yu Alison. GeneReach USA, Lexington; Estados Unidos Fil: Tsai, Chuan-Fu. GeneReach USA, Lexington; Estados Unidos Fil: Shen, Yu-Han. GeneReach USA, Lexington; Estados Unidos Fil: Tsai, Yun-Long. GeneReach USA, Lexington; Estados Unidos Fil: Chang, Hsiao-Fen G. GeneReach USA, Lexington; Estados Unidos Fil: Wang, Hwa-Tang Thomas. GeneReach USA, Lexington; Estados Unidos Fil: Balasuriya, Udeni B.R. Louisiana State University. School of Veterinary Medicine. Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences; Estados Unidos 2019-02-21T15:49:47Z 2019-02-21T15:49:47Z 2018 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/4481 https://www.onlinelibrary.wiley.com/doi/10.1111/evj.13032 0425-1644 https://doi.org/10.1111/evj.13032 eng info:eu-repo/semantics/restrictedAccess application/pdf Wiley Equine veterinary journal (24 October 2018) |
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Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing |
spellingShingle |
Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing Cook, R.F. Barrandeguy, Maria Edith Lee, Pei-Yu Alison Tsai, Chuan-Fu Shen, Yu-Han Tsai, Yun-Long Chang, Hsiao-Fen G. Wang, Hwa-Tang Thomas Balasuriya, Udeni B.R. Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
description |
Background: Control of equine infectious anaemia (EIA) currently depends on serological diagnosis of infected equids. However, recently infected
equids may not produce detectable anti-EIAV antibodies up to 157 days post infection and so present a high transmission risk. Therefore, direct nucleic
acid detection methods are urgently needed to improve EIAV surveillance and management programs in counties where the disease is endemic.
Objectives: To evaluate a field-deployable, reverse transcription-insulated isothermal PCR (RT-iiPCR) assay targeting the conserved 50 untranslated
region (50 UTR)/exon 1 of the tat gene of EIAV.
Study design: The analytical and clinical performance of the newly developed EIAV RT-iiPCR was evaluated by comparison with a EIAV real-time RTPCR
(RT-qPCR) along with the AGID test.
Methods: Analytical sensitivity was determined using in vitro transcribed RNA containing the target area of the 50 UTR/tat gene and samples from two
EIAV-positive horses. Specificity was verified using nine common equine viruses. Clinical performance was evaluated by comparison with EIAV RT-qPCR
and AGID using samples derived from 196 inapparent EIAV carrier horses.
Results: EIAV RT-iiPCR did not react with other commonly encountered equine viruses and had equivalent sensitivity (95% detection limit of eight
genome equivalents), with a concordance of 95.41% to conventional EIAV RT-qPCR. However, the RT-qPCR and RT-iiPCR had sensitivities of 43.75 and
50.00%, respectively, when compared to the AGID test.
Main limitations: Low viral loads commonly encountered in inapparent EIAV carriers may limit the diagnostic sensitivity of RT-PCR-based tests.
Conclusions: Although EIAV RT-iiPCR is not sufficiently sensitive to replace the current AGID test, it can augment control efforts by identifying recently
exposed or “serologically silent” equids, particularly as the latter often represent a significant transmission risk because of high viral loads. Furthermore,
the relatively low cost and field-deployable design enable utilisation of EIAV RT-iiPCR even in remote regions. |
format |
info:ar-repo/semantics/artículo |
topic_facet |
Caballos Virus de los Animales Horses Equine Infectious Anaemia Diagnosis Diagnóstico Anemia Infecciosa Equina PCR Animal Viruses Equinos Insulated Isothermal RT-PCR Point-of-Need Testing |
author |
Cook, R.F. Barrandeguy, Maria Edith Lee, Pei-Yu Alison Tsai, Chuan-Fu Shen, Yu-Han Tsai, Yun-Long Chang, Hsiao-Fen G. Wang, Hwa-Tang Thomas Balasuriya, Udeni B.R. |
author_facet |
Cook, R.F. Barrandeguy, Maria Edith Lee, Pei-Yu Alison Tsai, Chuan-Fu Shen, Yu-Han Tsai, Yun-Long Chang, Hsiao-Fen G. Wang, Hwa-Tang Thomas Balasuriya, Udeni B.R. |
author_sort |
Cook, R.F. |
title |
Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
title_short |
Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
title_full |
Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
title_fullStr |
Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
title_full_unstemmed |
Rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal RT-PCR assay to aid diagnosis under field conditions |
title_sort |
rapid detection of equine infectious anaemia virus nucleic acid by insulated isothermal rt-pcr assay to aid diagnosis under field conditions |
publisher |
Wiley |
publishDate |
2018 |
url |
http://hdl.handle.net/20.500.12123/4481 https://www.onlinelibrary.wiley.com/doi/10.1111/evj.13032 https://doi.org/10.1111/evj.13032 |
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