Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material
A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasm...
| Autores principales: | , , , |
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| Formato: | article |
| Lenguaje: | Inglés |
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American Society for Microbiology ("ASM")
2022
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| Acceso en línea: | http://hdl.handle.net/20.500.11939/8057 https://journals.asm.org/doi/epub/10.1128/AEM.66.5.2071-2078.2000 |
| _version_ | 1855032721396465664 |
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| author | Llop, Pablo Bonaterra, Anna Penyalver, Javier López, María M. |
| author_browse | Bonaterra, Anna Llop, Pablo López, María M. Penyalver, Javier |
| author_facet | Llop, Pablo Bonaterra, Anna Penyalver, Javier López, María M. |
| author_sort | Llop, Pablo |
| collection | ReDivia |
| description | A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity. |
| format | article |
| id | ReDivia8057 |
| institution | Instituto Valenciano de Investigaciones Agrarias (IVIA) |
| language | Inglés |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | American Society for Microbiology ("ASM") |
| publisherStr | American Society for Microbiology ("ASM") |
| record_format | dspace |
| spelling | ReDivia80572025-04-25T14:48:42Z Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material Llop, Pablo Bonaterra, Anna Penyalver, Javier López, María M. Nested-PCR Sensitivity H20 Plant diseases U30 Research methods Erwinia amylovora Diagnostic techniques Quarantine A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity. 2022-04-29T16:15:31Z 2022-04-29T16:15:31Z 2000 article publishedVersion Llop, P., Bonaterra, A., Peñalver, J. & López, M. M. (2000). Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Applied and Environmental Microbiology, 66(5), 2071-2078. 0099-2240 (print ISSN) 1098-5336 (eISSN) http://hdl.handle.net/20.500.11939/8057 10.1128/AEM.66.5.2071-2078.2000 https://journals.asm.org/doi/epub/10.1128/AEM.66.5.2071-2078.2000 en Funding: Subdirección General de Sanidad Vegetal, MAPA, Madrid, Spain, CICYT project AGF98 0402CO302, and SMT project 4-CT98 2252 for Atribución-NoComercial-SinDerivadas 3.0 España http://creativecommons.org/licenses/by-nc-nd/3.0/es/ openAccess American Society for Microbiology ("ASM") electronico |
| spellingShingle | Nested-PCR Sensitivity H20 Plant diseases U30 Research methods Erwinia amylovora Diagnostic techniques Quarantine Llop, Pablo Bonaterra, Anna Penyalver, Javier López, María M. Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title | Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title_full | Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title_fullStr | Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title_full_unstemmed | Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title_short | Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material |
| title_sort | development of a highly sensitive nested pcr procedure using a single closed tube for detection of erwinia amylovora in asymptomatic plant material |
| topic | Nested-PCR Sensitivity H20 Plant diseases U30 Research methods Erwinia amylovora Diagnostic techniques Quarantine |
| url | http://hdl.handle.net/20.500.11939/8057 https://journals.asm.org/doi/epub/10.1128/AEM.66.5.2071-2078.2000 |
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