1 Development of PCR-hybridization for the identification of major Gram positive bacteria causing bacteremia. Athicha Mahayotha 1 , Rosarin Wongvilairat 1 , Surang Dejsirilert 2 , Tawatchai Sumpradit 1 , Anusak Kerdsin 2 . 1 Department of Medical Science, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand. 2 National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand. E-mail address: aroon59@hotmail.com Abstract Blood culture is considered to be a gold standard for diagnosis of bacterial septicemia. However, this method has some limitations. Molecular techniques for detection of DNA of bacteria causing septicemia such as PCR and hybridization are sensitive, specific and more rapid compared with blood culture. The aims of this project are to develop PCR – slot blot hybridization technique for detection and identification of Gram positive bacteria causing bacterimia. 16S rRNA gene of gram positive cocci (11 isolates) were extracted and amplified by using universal primer. These fragments of each species were cloned in plasmids and preserved as clone libraries. Plasmid DNA was amplified by using M13 primer and PCR product used as DNA template to blot on a nylon membrane. V6 region in 16S rRNA gene of Streptococcus pneumoniae was amplified by PCR using digoxigenin-11-dUTP. PCR product was an initial experimental probe to optimize conditions of the hybridization with the reference DNA blotted on nylon membrane. Various anneal temperature and concentration of SSC buffer were tested to determine optimum hybridization conditions based on visual observation by using anti-DIG-AP conjugate, CDP-Star TM . This study found that the hybridize temperature at 65 o C exhibited more specific for probe of S. aureus, S. epidermidis and E. faecalis and unspecific for Streptococci. At 67 o C, hybridization exhibited more intense signal when used probe of all streptococcal species without any cross hybridization. The optimal condition of PCR-Slot blot Hybridization that was chosen as 67 o C of the hybridize temperature using for method validation. The lowest of pure DNA concentration of S. aureus ATCC 25923 which the PCR-Slot blot hybridization was given as positive results, was 0.7ng/μl. Sensitivity and specificity of PCR-Slot blot hybridization were highly value (100%). The results revealed that probe design and the optimized conditions were successful in the detection of gram positive septicemia-bacteria. Key words: Septicemia, Cultural method, PCR, Hybridization, 16S rRNA gene.