Development and validation of an improved PCR method using the 23S-5S intergenic spacer for detection of rickettsiae in dermacentor variabilis ticks and tissue samples from humans and laboratory animals

Details

• Personal Author:
  Apperson CS ; Kakumanu ML ; Meshnick SR ; Nicholson WL ; Ponnusamy L ; Sutton HT
• Description:
  A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia-specific PCR targets (ompA, gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "Candidatus Rickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. "Candidatus Rickettsia amblyommii," R. montanensis, R. felis, and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii. [Description provided by NIOSH]
• Subjects:
  Biological Assay Genetics Laboratories Microbiology Nucleotides Occupational Health Safety
• Keywords:
  Bacteria Bacterial-disease Bacterial Cultures Bacterial Infections Bioassays Deoxyribonucleic Acids Detectors Disease Vectors DNA Genes Genomics Humans Laboratory Animals Laboratory Techniques Nucleic Acids Parasitic Diseases Pathogens Proteins Sampling Sensitivity Testing Tickborne Diseases Tissue Culture

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• ISSN:
  0095-1137
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  Georgia ; North Carolina ; OSHA Region 4
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  54
• Issue:
  4
• NIOSHTIC Number:
  nn:20049094
• Citation:
  J Clin Microbiol 2016 Apr; 54(4):972-979
• Contact Point Address:
  Charles S. Apperson, Department of Entomology, North Carolina State University, Raleigh, North Carolina, USA
• Email:
  apperson@ncsu.edu
• Federal Fiscal Year:
  2016
• Performing Organization:
  University of North Carolina, Chapel Hill
• Peer Reviewed:
  True
• Start Date:
  20100901
• Source Full Name:
  Journal of Clinical Microbiology
• End Date:
  20150831
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:c9ec49e17845846f184227da9eb71dce7fe0de40b4f7ecbfb5f329e77d16269786a9e7d8ecdf7f7fde3c7f18405ca72d7fac4b039fe0240b9bc21379bfd85455
• Download URL:
  https://stacks.cdc.gov/view/cdc/203667/cdc_203667_DS1.pdf
• File Type:
  [PDF - 636.60 KB ]