Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil
Abstract Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnost...
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2021
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oai:doaj.org-article:535fabb9639a49db8a68d446357e20ca2021-11-14T12:19:55ZGenome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil10.1038/s41598-021-01196-42045-2322https://doaj.org/article/535fabb9639a49db8a68d446357e20ca2021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-01196-4https://doaj.org/toc/2045-2322Abstract Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.Ryan DomingoCristian PerezDiksha KlairHuong VuAlika Candelario-TochikiXupeng WangAmihan CamsonJaclyn Nicole UyMouauia SalamehDario ArizalaShefali DobhalGamze BolukJon-Paul BinghamFrancisco Ochoa-CoronaMd Emran AliJames P. StackJacqueline FletcherJenee OdaniDaniel JenkinsAnne M. AlvarezMohammad ArifNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021) |
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Medicine R Science Q Ryan Domingo Cristian Perez Diksha Klair Huong Vu Alika Candelario-Tochiki Xupeng Wang Amihan Camson Jaclyn Nicole Uy Mouauia Salameh Dario Arizala Shefali Dobhal Gamze Boluk Jon-Paul Bingham Francisco Ochoa-Corona Md Emran Ali James P. Stack Jacqueline Fletcher Jenee Odani Daniel Jenkins Anne M. Alvarez Mohammad Arif Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| description |
Abstract Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies. |
| format |
article |
| author |
Ryan Domingo Cristian Perez Diksha Klair Huong Vu Alika Candelario-Tochiki Xupeng Wang Amihan Camson Jaclyn Nicole Uy Mouauia Salameh Dario Arizala Shefali Dobhal Gamze Boluk Jon-Paul Bingham Francisco Ochoa-Corona Md Emran Ali James P. Stack Jacqueline Fletcher Jenee Odani Daniel Jenkins Anne M. Alvarez Mohammad Arif |
| author_facet |
Ryan Domingo Cristian Perez Diksha Klair Huong Vu Alika Candelario-Tochiki Xupeng Wang Amihan Camson Jaclyn Nicole Uy Mouauia Salameh Dario Arizala Shefali Dobhal Gamze Boluk Jon-Paul Bingham Francisco Ochoa-Corona Md Emran Ali James P. Stack Jacqueline Fletcher Jenee Odani Daniel Jenkins Anne M. Alvarez Mohammad Arif |
| author_sort |
Ryan Domingo |
| title |
Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| title_short |
Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| title_full |
Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| title_fullStr |
Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| title_full_unstemmed |
Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil |
| title_sort |
genome-informed loop-mediated isothermal amplification assay for specific detection of pectobacterium parmentieri in infected potato tissues and soil |
| publisher |
Nature Portfolio |
| publishDate |
2021 |
| url |
https://doaj.org/article/535fabb9639a49db8a68d446357e20ca |
| work_keys_str_mv |
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