title: [Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis].
abstract: Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR Toolbox assay were found to be 100%. The lowest detection limit of this method was determined as 100 genomic equivalent per PCR reaction mix. The new PCR kit is a rapid and accurate alternative to the conventional PCR methods since the toolbox includes all of the required chemicals, accessories and equipments. This ready-to-use PCR assay was appraised to be a valuable diagnostic tool for the detection of F.tularensis in the outbreak settings particularly in remote areas with limited resources.
authors: Public Health Institution of Turkey, National Tularemia Reference Laboratory, Ankara, Turkey. mdskilic2003@yahoo.com.
authors: Bekir
authors: B
authors: Celebi
authors: Selçuk
authors: S
authors: Kılıç
authors: Murat
authors: M
authors: Yeşilyurt
authors: Bülent
authors: B
authors: Acar
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D023482:Bioterrorism; D005604:Francisella tularensis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008074:Lipoproteins / Q000235:genetics; D008198:Lymph Nodes / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000592:standards*; D011237:Predictive Value of Tests; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014406:Tularemia / Q000175:diagnosis*; D014421:Turkey; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D008074:Lipoproteins; D011933:Reagent Kits, Diagnostic; C075214:TUL4 protein, Francisella tularensis