title: Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.
abstract: A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.
authors.affiliation: Naval Medical Research Center, Viral & Rickettsial Diseases Department, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA.
authors.forename: Ju
authors.initials: J
authors.lastname: Jiang
authors.forename: Brian J
authors.initials: BJ
authors.lastname: You
authors.forename: Evan
authors.initials: E
authors.lastname: Liu
authors.forename: Anisha
authors.initials: A
authors.lastname: Apte
authors.forename: Tamasin R
authors.initials: TR
authors.lastname: Yarina
authors.forename: Todd E
authors.initials: TE
authors.lastname: Myers
authors.forename: John S
authors.initials: JS
authors.lastname: Lee
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Francesconi
authors.forename: Monica L
authors.initials: ML
authors.lastname: O'Guinn
authors.forename: Nikoloz
authors.initials: N
authors.lastname: Tsertsvadze
authors.forename: Nino
authors.initials: N
authors.lastname: Vephkhvadze
authors.forename: Giorgi
authors.initials: G
authors.lastname: Babuadze
authors.forename: Ketevan
authors.initials: K
authors.lastname: Sidamonidze
authors.forename: Maka
authors.initials: M
authors.lastname: Kokhreidze
authors.forename: Marina
authors.initials: M
authors.lastname: Donduashvili
authors.forename: Tinatin
authors.initials: T
authors.lastname: Onashvili
authors.forename: Afrail
authors.initials: A
authors.lastname: Ismayilov
authors.forename: Nigar
authors.initials: N
authors.lastname: Agayev
authors.forename: Mubariz
authors.initials: M
authors.lastname: Aliyev
authors.forename: Nizam
authors.initials: N
authors.lastname: Muttalibov
authors.forename: Allen L
authors.initials: AL
authors.lastname: Richards
mesh_terms: D000818:Animals; D001382:Azerbaijan; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D003870:Dermacentor / Q000382:microbiology; D005846:Georgia (Republic); D006801:Humans; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012281:Rickettsia / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012282:Rickettsia Infections / Q000175:diagnosis* / Q000382:microbiology*; D012680:Sensitivity and Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes