I U H Khan1, M Cloutier1, M Libby1, D R Lapen1, G Wilkes1, E Topp2. 1. Ottawa Research and Development Centre (ORDC), Agriculture and Agri-Food Canada, Ottawa, ON, Canada. 2. London Research and Development Centre (LRDC), Agriculture and Agri-Food Canada, London, ON, Canada.
Abstract
AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg μl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.
AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg μl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.
Authors: Kaidi Wang; Lei Chen; Xiangyun Ma; Lina Ma; Keng C Chou; Yankai Cao; Izhar U H Khan; Greta Gölz; Xiaonan Lu Journal: Appl Environ Microbiol Date: 2020-10-01 Impact factor: 4.792
Authors: Matthew Zambri; Michel Cloutier; Zaky Adam; David R Lapen; Graham Wilkes; Mark Sunohara; Edward Topp; Guylaine Talbot; Izhar U H Khan Journal: BMC Microbiol Date: 2019-01-11 Impact factor: 3.605
Authors: Germán A Kopprio; Sucharit B Neogi; Harunur Rashid; Cecilia Alonso; Shinji Yamasaki; Boris P Koch; Astrid Gärdes; Rubén J Lara Journal: Front Microbiol Date: 2020-04-15 Impact factor: 5.640
Authors: Mary G Miltenburg; Michel Cloutier; Emilia Craiovan; David R Lapen; Graham Wilkes; Edward Topp; Izhar U H Khan Journal: BMC Microbiol Date: 2020-06-16 Impact factor: 3.605