PCR-based detection and identification of Burkholderia cepacia complex from clinical specimens

vPCR-BASED DETECTION AND IDENTIFICATION OFBURKHOLDERIA CEPACIA COMPLEX FROM CLINICAL SPECIMENS(PhD. Thesis)Dilek Hande BINNETGAZI UNIVERSITYINSTUTE OF SCIENCE AND TECHNOLOGYJanuary 2006ABSTRACTMolecular identification methods have gained importance at detection of bothPseudomonas genus and Burkholderia cepacia in recent years. During the research,molecular identification of B. cepacia complex from 97 clinical specimens werestudied. 770 base pair (bp) 16S rRNA gene region specific for B. cepacia complexhad been amplified by PCR. The PCR products of B. cepacia complex wereverified using, TaqI, RsaI and Tsp5091 restriction endonucleases enzymes. In thisstudy, determination of nucleotide sequence of strains which were verified withrestriction endonucleases enzymes had been aimed and DNA sequence analysis ofspecimens were applied. The nucleotide sequence which was defined as a result ofDNA sequence analysis of verified B. cepacia complex isolates compared withB. cepacia complex (AY207313) nucleotide sequence which registered in GenBank.At the end of comparison, nucleotide sequences, was found to carry the samesequence with the previously submitted sequence. As consequence of AP-PCRmethod which was used for molecular typing of 28 B. cepacia complex isolates andvithe specimens are separated into 6 different bands. In order to determine thenumbers and greatness of bands which caused by amplifying fragments, DNAbioanalysis process was applied to the fragments. At the end of this analysis it wasdetected that same fragments are separated into 11 different groups. According toour study results, molecular methods in B. cepacia complex identification wereconfirmed and verified as more confident and faster tests. Due to thesespecifications molecular methods are also advised in addition to traditional culturemethods for clinical research laboratories.Science Code : 203.1.104Key Words : Burkholderia cepacia complex, identification, DNA bioanalysis,PCR, AP-PCRPage Number : 84Adviser : Prof. Dr. Güven Uraz