| Autor: |
Blanton LS; 1Division of Infectious Diseases, Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas., Quade BR; 1Division of Infectious Diseases, Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas., Bouyer DH; 2Department of Pathology, The University of Texas Medical Branch, Galveston, Texas. |
| Jazyk: |
angličtina |
| Zdroj: |
Vector borne and zoonotic diseases (Larchmont, N.Y.) [Vector Borne Zoonotic Dis] 2019 Aug; Vol. 19 (8), pp. 637-639. Date of Electronic Publication: 2019 Apr 25. |
| DOI: |
10.1089/vbz.2018.2428 |
| Abstrakt: |
Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis ) have been discovered- Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis -like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme Nla IV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of Nla IV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with Nla IV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis , and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
