Improvement of the real-time polymerase chain reaction method used in the diagnosis of brucellosis

Brucellosis; is a zoonosis with high morbidity and low-mortality which can cause various clinical findings, relapses and complications around the world. Although the true incidence of Brucella infection in humans is not exactly known, it is reported that the incidence is between <0.01 and >0.002 in endemic areas. Routine diagnosis of brucellosis is often performed by culture and serology. However, the sensitivity of culture methods may vary depending on the disease stage, Brucella species, culture medium and blood culture technique. In addition, the specificity of serological methods is low due to antibodies against cross-reactive microorganisms. Polymerase Chain Reaction (PCR) based approaches have gained importance to diagnose because of these adversities in conventional diagnostic tests. These methods are good alternative in the rapid and reliable diagnosis of human brucellosis. In this study, we aimed to develop a rapid, specific, sensitive and reliable multiplex Real-time Polymerase Chain Reaction (mRt-PCR) method that can be used in the early diagnosis of brucellosis and to compare this method with conventional diagnostic tests. The lowest detection limit of the mRt-PCR method was investigated in simulated serum and whole blood samples containing Brucella melitensis ATCC 23456 standard strain with different concentration varying between 108 and 101 cfu / ml. These samples firstly were extracted with Thermo and Norgen DNA isolation kits. Then the lowest detection limit of the method was determined by using Maxima SYBR green, QuantiTect and Ampliqon Tempase PCR master mixes in Rotor-Gene (Corbett RG 6000, Australia) and Bio-Rad (CFX-96 C1000 Touch Real-time system) thermal cycler devices. Validation studies of the optimized method were performed, then the efficacy of the method was evaluated on 21 blood culture bottles with positive reproductive signal and 57 serum samples of brucellosis suspicious patients. The lowest detection limit of the method was found 104 cfu / ml in both thermal cycler when the Maxima SYBR green 2x master mix was used. The amplification was provided at a concentration of 102 cfu / ml in the reactions that set up with the probe-based QuantiTect and Ampliqon Tempase master mixes. The validation studies of the optimized method was performed in serum samples with Thermo DNA extraction kit-Ampliqon master mix. It has been determined that the method showed high level accuracy, precision, specificity and sensitivity by performing the validation studies. Positive mRt-PCR results were detected in 33 (57.8%) serum samples of 57 patients. Only the 41 patients had blood culture samples and Brucella spp. was isolated from 21 of them. Moreover, serum PCR results of all Brucella positive blood culture samples were positive as Brucella melitensis. Additionally; serum PCR positivity was recorded in 12 of 20 patients whose blood culture was negative and at least one of the serologic tests showed significant positive results (STAT ≥1 / 160 and / or Coombs titer ≥1 / 320). These patients are diagnosed as ''presumptive case'' according to the CDC laboratory criteria for diagnosis of brucellosis. Our method can be used safely in the diagnosis of brucellosis from both serum and whole blood samples or other clinical specimens and blood culture bottles. Key Words: Brucellosis, B. melitensis, Molecular assay, Real-time PCR, Optimization, Serology