Development of simplex and multiplex pcr protocols for the identification of abortive zoonotic bacteria in women

Zoonotic bacteria can be considered as important infectious agents that can cause miscarriage in women. Microbial agents that cause miscarriage in women have similar symptoms seen in other diseases. This situation reveals the necessity to evaluate all factors thought to cause the disease. Investigating each factor individually is difficult, time consuming and costly, it requires biosafety practices and technical skills. For this reason, molecular methods that provide multiple analyzes are preferred because of the false positivity in serological tests of the disease in the acute period. It was designed primer pairs specifically for the genes squence of Brucella abortus (IS711, omp2b), Coxiella burnetii (com1, IS1111a), Chlamydia abortus (ompA, pmp 90/91) and Listeria monocytogenes (hly, iap). These primer pairs were designed specifically for this study, first time. Firstly, simplex PCR studies were carried out with the primer pairs selected for each organism then multiplex PCR studies including duplex and triplex in different double and triple combinations were carried out with the primer pairs selected for each organism. The sensitivity and specificity of the primers selected as a result of amplification studies with different DNA concentrations were qualitatively determined. Primer pairs; BaısF2/BaısR2 (304 bp) for B. abortus, CbısF3/CbısR3 (430 bp) for C. burnetii, CapmpF3/CapmpR3 (588 bp) for C. abortus and LmhlyF1/LmhlyR1 (526 bp) for L. monocytogenes were showed high sensitivity and specificity. Duplex and triplex PCR studies were carried out to all combinations with these primer pairs. As a result, clear amplification of the relevant genes sequences in the presence of 10-1 concentration of template DNA was observed. Multiplex PCR protocol performed with primers specially designed for our study will allow simultaneous diagnosis of Brucella abortus, Coxiella burnetii, Chlamydia abortus and Listeria monocytogenes species.