Cargando…
Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants
SIMPLE SUMMARY: Contagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the princ...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143204/ https://www.ncbi.nlm.nih.gov/pubmed/32204335 http://dx.doi.org/10.3390/ani10030509 |
_version_ | 1783519559427293184 |
---|---|
author | Tumino, Serena Tolone, Marco Parco, Alessio Puleio, Roberto Arcoleo, Giuseppe Manno, Claudia Nicholas, Robin A.J. Loria, Guido Ruggero |
author_facet | Tumino, Serena Tolone, Marco Parco, Alessio Puleio, Roberto Arcoleo, Giuseppe Manno, Claudia Nicholas, Robin A.J. Loria, Guido Ruggero |
author_sort | Tumino, Serena |
collection | PubMed |
description | SIMPLE SUMMARY: Contagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the principal etiological agent of CA, especially in sheep. Clinical signs are characterised by mastitis, arthritis, keratoconjunctivitis and occasionally, abortion. Rapid, accurate and cost-effective field tests are urgently needed for effective control of M. agalactiae mastitis. Our study illustrated the validation of a Loop-Mediated Isothermal Amplification (LAMP) test for the detection of M. agalactiae in dairy sheep in order to confirm its application as a diagnostic tool in the field level. ABSTRACT: Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 10(3) CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA. |
format | Online Article Text |
id | pubmed-7143204 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-71432042020-04-14 Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants Tumino, Serena Tolone, Marco Parco, Alessio Puleio, Roberto Arcoleo, Giuseppe Manno, Claudia Nicholas, Robin A.J. Loria, Guido Ruggero Animals (Basel) Communication SIMPLE SUMMARY: Contagious agalactia (CA) is an infectious disease of small ruminants endemic in the Mediterranean countries, causing significant socioeconomic impacts predominantly on small-scale farmers who still subsist on marginal lands. Mycoplasma agalactiae is historically considered the principal etiological agent of CA, especially in sheep. Clinical signs are characterised by mastitis, arthritis, keratoconjunctivitis and occasionally, abortion. Rapid, accurate and cost-effective field tests are urgently needed for effective control of M. agalactiae mastitis. Our study illustrated the validation of a Loop-Mediated Isothermal Amplification (LAMP) test for the detection of M. agalactiae in dairy sheep in order to confirm its application as a diagnostic tool in the field level. ABSTRACT: Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 10(3) CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84–0.96) positive milk samples compared to 69 (77%, 95%CI 0.59–0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA. MDPI 2020-03-19 /pmc/articles/PMC7143204/ /pubmed/32204335 http://dx.doi.org/10.3390/ani10030509 Text en © 2020 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 | Communication Tumino, Serena Tolone, Marco Parco, Alessio Puleio, Roberto Arcoleo, Giuseppe Manno, Claudia Nicholas, Robin A.J. Loria, Guido Ruggero Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title | Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title_full | Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title_fullStr | Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title_full_unstemmed | Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title_short | Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants |
title_sort | validation of loop-mediated isothermal amplification (lamp) field tool for rapid and sensitive diagnosis of contagious agalactia in small ruminants |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143204/ https://www.ncbi.nlm.nih.gov/pubmed/32204335 http://dx.doi.org/10.3390/ani10030509 |
work_keys_str_mv | AT tuminoserena validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT tolonemarco validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT parcoalessio validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT puleioroberto validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT arcoleogiuseppe validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT mannoclaudia validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT nicholasrobinaj validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants AT loriaguidoruggero validationofloopmediatedisothermalamplificationlampfieldtoolforrapidandsensitivediagnosisofcontagiousagalactiainsmallruminants |