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Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification

Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection...

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Autores principales: Manjunatha, C., Sharma, Sapna, Kulshreshtha, Deepika, Gupta, Sangeeta, Singh, Kartar, Bhardwaj, Subhash C., Aggarwal, Rashmi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919678/
https://www.ncbi.nlm.nih.gov/pubmed/29698484
http://dx.doi.org/10.1371/journal.pone.0196409
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author Manjunatha, C.
Sharma, Sapna
Kulshreshtha, Deepika
Gupta, Sangeeta
Singh, Kartar
Bhardwaj, Subhash C.
Aggarwal, Rashmi
author_facet Manjunatha, C.
Sharma, Sapna
Kulshreshtha, Deepika
Gupta, Sangeeta
Singh, Kartar
Bhardwaj, Subhash C.
Aggarwal, Rashmi
author_sort Manjunatha, C.
collection PubMed
description Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA(68)) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65(o) C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in gel electrophoresis. Our assay is significantly faster than the conventional methods used in the identification of P. triticina. The assay developed in the study shall be very much useful in the development of diagnostic kit for monitoring disease, creation of prediction model and efficient management of disease.
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spelling pubmed-59196782018-05-11 Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification Manjunatha, C. Sharma, Sapna Kulshreshtha, Deepika Gupta, Sangeeta Singh, Kartar Bhardwaj, Subhash C. Aggarwal, Rashmi PLoS One Research Article Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA(68)) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65(o) C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in gel electrophoresis. Our assay is significantly faster than the conventional methods used in the identification of P. triticina. The assay developed in the study shall be very much useful in the development of diagnostic kit for monitoring disease, creation of prediction model and efficient management of disease. Public Library of Science 2018-04-26 /pmc/articles/PMC5919678/ /pubmed/29698484 http://dx.doi.org/10.1371/journal.pone.0196409 Text en © 2018 Manjunatha et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Manjunatha, C.
Sharma, Sapna
Kulshreshtha, Deepika
Gupta, Sangeeta
Singh, Kartar
Bhardwaj, Subhash C.
Aggarwal, Rashmi
Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title_full Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title_fullStr Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title_full_unstemmed Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title_short Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification
title_sort rapid detection of puccinia triticina causing leaf rust of wheat by pcr and loop mediated isothermal amplification
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919678/
https://www.ncbi.nlm.nih.gov/pubmed/29698484
http://dx.doi.org/10.1371/journal.pone.0196409
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