Enhanced Single‐tube Multiplex PCR Assay for Detection and Identification of SixArcobacterSpecies
| Autor: | Izhar U. H. Khan, Mark Libby, Michel Cloutier, Edward Topp, Graham Wilkes, David R. Lapen |
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| Rok vydání: | 2017 |
| Předmět: |
DNA
Bacterial 0301 basic medicine 030106 microbiology Water source Sensitivity and Specificity Applied Microbiology and Biotechnology Microbiology Single tube 03 medical and health sciences Species Specificity Multiplex polymerase chain reaction Animals Humans Arcobacter biology General Medicine biology.organism_classification rpoB Molecular biology Arcobacter species Restriction fragment length polymorphism Primer (molecular biology) Gram-Negative Bacterial Infections Multiplex Polymerase Chain Reaction Biotechnology |
| Zdroj: | Journal of Applied Microbiology. 123:1522-1532 |
| ISSN: | 1365-2672 1364-5072 |
| DOI: | 10.1111/jam.13597 |
| Popis: | 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 target 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 fecal 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 for mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for etiological and epidemiological studies. This article is protected by copyright. All rights reserved. |
| Databáze: | OpenAIRE |
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