Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR
‘<i>Candidatus</i> Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in <i>Solanaceae</i> and <i>Apiaceae</i> crops. CaLsol detection in psyllids often requires insect destruction, preventi...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-05-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/6/1104 |
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| author | María Quintana Leandro de-León Jaime Cubero Felipe Siverio |
| author_facet | María Quintana Leandro de-León Jaime Cubero Felipe Siverio |
| author_sort | María Quintana |
| collection | DOAJ |
| description | ‘<i>Candidatus</i> Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in <i>Solanaceae</i> and <i>Apiaceae</i> 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 <i>Bactericera trigonica</i> (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<sup>TM</sup>, HotSHOT, and DNeasy<sup>®</sup>). 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 <i>B. trigonica</i> 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. |
| first_indexed | 2024-03-09T22:59:40Z |
| format | Article |
| id | doaj.art-0741f8353fad4ce5a742422f2a6494cc |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T22:59:40Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-0741f8353fad4ce5a742422f2a6494cc2023-11-23T18:03:03ZengMDPI AGMicroorganisms2076-26072022-05-01106110410.3390/microorganisms10061104Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCRMaría Quintana0Leandro de-León1Jaime Cubero2Felipe Siverio3Unidad de Protección Vegetal, Instituto Canario de Investigaciones Agrarias, 38270 San Cristóbal de La Laguna, SpainDepartamento de Bioquímica, Microbiología, Biología Celular y Genética, Facultad de Farmacia, Universidad de La Laguna, 38200 San Cristóbal de La Laguna, SpainCentro Nacional Instituto de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), 28040 Madrid, SpainUnidad de Protección Vegetal, Instituto Canario de Investigaciones Agrarias, 38270 San Cristóbal de La Laguna, Spain‘<i>Candidatus</i> Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in <i>Solanaceae</i> and <i>Apiaceae</i> 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 <i>Bactericera trigonica</i> (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<sup>TM</sup>, HotSHOT, and DNeasy<sup>®</sup>). 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 <i>B. trigonica</i> 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.https://www.mdpi.com/2076-2607/10/6/1104vectordiseasebacterium<i>Bactericera trigonica</i>HLBethanol contamination |
| spellingShingle | María Quintana Leandro de-León Jaime Cubero Felipe Siverio Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR Microorganisms vector disease bacterium <i>Bactericera trigonica</i> HLB ethanol contamination |
| title | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR |
| title_full | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR |
| title_fullStr | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR |
| title_full_unstemmed | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR |
| title_short | Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘<i>Candidatus</i> Liberibacter Solanacearum’ by qPCR |
| title_sort | assessment of psyllid handling and dna extraction methods in the detection of i candidatus i liberibacter solanacearum by qpcr |
| topic | vector disease bacterium <i>Bactericera trigonica</i> HLB ethanol contamination |
| url | https://www.mdpi.com/2076-2607/10/6/1104 |
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