Journal Article

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Stack, James P., Ochoa-Corona, Francisco, Arif, Mohammad, Uy, Jaclyn Nicole, Camson, Amihan, Alvarez, Anne M., Vu, Huong, Wang, Xupeng, Jenkins, Daniel, Boluk, Gamze, Bingham, Jon-Paul, Domingo, Ryan, Klair, Diksha, Candelario-Tochiki, Alika, Arizala, Dario, Ali, Md Emran, Dobhal, Shefali, Odani, Jenee, Salameh, Mouauia, Fletcher, Jacqueline, Perez, Cristian

2021

Overview

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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/326/1320 / 631/326/41 / 631/326/421 / Biosecurity / Computer Simulation / Deoxyribonucleic acid / DNA / DNA, Bacterial - genetics / Epidemiology / Field tests / Gene amplification / Genome, Bacterial / Genomes / Humanities and Social Sciences / Limit of Detection / Molecular Diagnostic Techniques - methods / multidisciplinary / Nucleic Acid Amplification Techniques - methods / Operators / Pectobacterium / Pectobacterium - genetics / Pectobacterium - isolation & purification / Potatoes / Reproducibility of Results / Science / Science (multidisciplinary) / Soft rot / Soil Microbiology / Solanum tuberosum - microbiology / Strains (organisms)