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Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR
‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this wor...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9230594/ https://www.ncbi.nlm.nih.gov/pubmed/35744622 http://dx.doi.org/10.3390/microorganisms10061104 |
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author | Quintana, María de-León, Leandro Cubero, Jaime Siverio, Felipe |
author_facet | Quintana, María de-León, Leandro Cubero, Jaime Siverio, Felipe |
author_sort | Quintana, María |
collection | PubMed |
description | ‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsure(TM), HotSHOT, and DNeasy(®)). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study. |
format | Online Article Text |
id | pubmed-9230594 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-92305942022-06-25 Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR Quintana, María de-León, Leandro Cubero, Jaime Siverio, Felipe Microorganisms Article ‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsure(TM), HotSHOT, and DNeasy(®)). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study. MDPI 2022-05-26 /pmc/articles/PMC9230594/ /pubmed/35744622 http://dx.doi.org/10.3390/microorganisms10061104 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Quintana, María de-León, Leandro Cubero, Jaime Siverio, Felipe Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title_full | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title_fullStr | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title_full_unstemmed | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title_short | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR |
title_sort | assessment of psyllid handling and dna extraction methods in the detection of ‘candidatus liberibacter solanacearum’ by qpcr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9230594/ https://www.ncbi.nlm.nih.gov/pubmed/35744622 http://dx.doi.org/10.3390/microorganisms10061104 |
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