title: Pyrosequencing Using SL and 5S rRNA as Molecular Markers for Identifying Zoonotic Filarial Nematodes in Blood Samples and Mosquitoes.
abstract: Lymphatic filariasis is principally caused by Wuchereria bancrofti, and Brugia malayi. The other two filarial nematode species, Brugia pahangi and Dirofilaria immitis, possibly cause human zoonotic diseases.
  
  
  
  We propose the development of a PCR assay linked with DNA pyrosequencing as a rapid tool to identify W. bancrofti, B. malayi, B. pahangi, and D. immitis in blood samples and mosquitoes. Primers targeting the fragment of the 5S ribosomal RNA and spliced leader sequences were newly designed and developed to identify these four filarial nematodes. Analytical sensitivity and specificity were evaluated.
  
  
  
  Pyrosequencing determination of nucleotide variations within 36 nucleotides for B. malayi and B. pahangi, and 32 nucleotides for W. bancrofti and D. immitis is sufficient for differentiation of those filarial nematodes, and for detection of intraspecies genetic variation of B. malayi. This analysis could detect a single B. malayi, B. pahangi, W. bancrofti, and D. immitis microfilaria in blood samples.
  
  
  
  Overall, the PCR-linked pyrosequencing-based method was faster than direct sequencing and less expensive than real-time PCR or direct sequencing. This is the possibility of choice that can be applied in a high-throughput platform for identification and surveillance of reservoirs and vectors infected with lymphatic filaria in endemic areas.
authors.affiliation: 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand .|2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .|3 Faculty of Medicine, Mahasarakham University , Mahasarakham, Thailand .
authors.forename: Oranuch
authors.initials: O
authors.lastname: Sanpool
authors.affiliation: 2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .|4 Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University , Pathum Thani, Thailand .
authors.forename: Chairat
authors.initials: C
authors.lastname: Tantrawatpan
authors.affiliation: 2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .|3 Faculty of Medicine, Mahasarakham University , Mahasarakham, Thailand .
authors.forename: Tongjit
authors.initials: T
authors.lastname: Thanchomnang
authors.affiliation: 2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .|5 Department of Medical Technology, School of Allied Health Sciences and Public Health, Walailak University , Nakhon Si Thammarat, Thailand .
authors.forename: Penchom
authors.initials: P
authors.lastname: Janwan
authors.affiliation: 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand .|2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .
authors.forename: Pewpan M
authors.initials: PM
authors.lastname: Intapan
authors.affiliation: 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand .|2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .
authors.forename: Rutchanee
authors.initials: R
authors.lastname: Rodpai
authors.affiliation: 2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .|6 Department of Microbiology, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand .
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
authors.affiliation: 7 Parasitology Unit, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University , Bangkok, Thailand .
authors.forename: Piyanan
authors.initials: P
authors.lastname: Taweethavonsawat
authors.affiliation: 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University , Khon Kaen, Thailand .|2 Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University , Khon Kaen, Thailand .
authors.forename: Wanchai
authors.initials: W
authors.lastname: Maleewong
mesh_terms: D000330:Aedes / Q000469:parasitology; D000818:Animals; D015415:Biomarkers; D002009:Brugia / Q000302:isolation & purification*; D002371:Cat Diseases / Q000469:parasitology; D002415:Cats; D004183:Dirofilaria immitis / Q000302:isolation & purification*; D004283:Dog Diseases / Q000469:parasitology; D004285:Dogs; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D018510:RNA, Helminth / Q000302:isolation & purification*; D016053:RNA, Protozoan / Q000235:genetics; D012680:Sensitivity and Specificity; D014958:Wuchereria bancrofti / Q000302:isolation & purification*; D015047:Zoonoses
chemical_list: D015415:Biomarkers; D018510:RNA, Helminth; D016053:RNA, Protozoan
keywords: Diagnostics; Filaria; Genetics; PCR; Parasitology