Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis.

Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 101 to 108 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe-based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 104 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 103 cfu/mL and 102 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe-based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.

Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis.

OBJECTIVE: Brucellosis is a zoonotic disease with various clinical presentations and early diagnosis is crucial to avoid severe complications. Due to limitations of conventional diagnostic methods, polymerase chain reaction (PCR) based approaches have gained importance in diagnosis.We aimed to evaluate diagnostic value of multiplex real time-PCR (mRT-PCR) in serum samples collected from brucellosis suspected patients by comparision sensitivity of mRT-PCR with those of conventional diagnostic methods. MATERIAL AND METHODS: A total of 249 serum samples collected from the suspected brucellosis patients admitted to the hospitals in three different provinces were analyzed by serological tests, culture and mRT-PCR. In laboratories of the participating hospital, serum samples were tested for the Brucella specific antibody by commercial serological kits including standart tube agglutination test (STAT), Coombs' test, and immunocapture test (ICT). Blood culture was performed for 153 of the patients in the participating hospital. All serum samples were analyzed for the presence of Brucella DNA by mRT-PCR. RESULTS: According to laboratory test results, 215 of the 249 suspected cases having comparible clinical data were identified as brucellosis cases. Of the 215 brucellosis cases, 36 were diagnosed as definitive cases, the remaning 179 patients were presumptive cases. Sensitivity of mRT-PCR in the samples that were positive by ICT, STAT, Coombs' test, and blood culture was 70.2%, 77.3%, 83%, and 97.2%, respectively. By using mRT-PCR, additional 17 suspected patients were diagnosed as presumptive cases. Among the mRT-PCR positive serum samples, Brucella abortus was detected in 3 samples (1.9%), the remaining 156 samples (98.1%) had B. melitensis DNA. CONCLUSION: Our results indicate that mRT-PCR can be considered a useful diagnostic tool in patients who have negative serologic test results, and in detection of Brucella species.

[Comparison of two commercial DNA extraction kits and PCR master mixes for the detection of Brucella from blood samples and blood culture bottles]. / Kandan ve kan kültür siselerinden Brucella saptanmasinda iki farkli ticari DNA ekstraksiyon kiti ve PCR master kiksinin karsilastirilmasi.

The diagnosis of human brucellosis requires culture or serological tests for the conformation of the clinical findings. Isolation of the bacteria is used as a gold standard, however it is time consuming and processing of positive cultures has a potential risk for laboratory acquired human brucellosis. Polymerase chain reaction (PCR) based methods have offered new approaches for early diagnosis of brucellosis and reduce the risk of laboratory acquired human brucellosis. A major limitation of the PCR method is the difficulty to remove the inhibitors in specimens. The aim of this study was to determine the performance of two DNA extraction kits by using two separate PCR master mixes and to determine appropriate "extraction kit - PCR master mix" combination for the diagnosis of Brucella from whole blood samples and blood culture bottles. Two commercial DNA extraction kits, NORGEN Blood DNA isolation kit (Norgen) and Thermo Scientific GeneJet Whole blood genomic DNA purification kit (Thermo Fisher Scientific, USA) and two PCR master mixes, QuantiTect multiplex PCR (QuentiTect, Qiager, Almanya) and Ampliqon Multiplex TEMPase (Amliqon, Denmark) were assessed on 30 simulated blood samples with known concentrations (102-104 cfu/ml) of Brucella melitensis ATCC 23456 strain and 10 blood culture bottles that gave positive signal. By using different combinations of extraction kits and PCR master mixes, a total of 160 different multiplex real-time PCR (Rt-PCR) trials were performed with probes and primers specific to Brucella spp., B.melitensis, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the 120 Rt-PCR trials performed on the DNA samples extracted from blood samples gave positive results with GAPDH probe/primers. The rate of positive PCR results for Brucella spp. was 96.7% for the combination of Norgen-QuantiTect, 93.3% for Thermo-Ampliqon, 93.3% for Thermo-QuantiTect, and 86.7% for Norgen-Ampliqon. The frequency of positive B.melitensis results for these combinations were 96.7%, 93.3%, 56.7% and 90%, respectively. In the samples with the bacterial density of 102 cfu/ml, Brucella spp. detection rates were 80% for Thermo-Ampliqon and Norgen-Ampliqon, and 90% for Thermo-QuantiTect and Norgen-QuantiTect; and for B.melitensis positivite rates were 90%, 70%, 20%, and 90%, respectively. Rt-PCR assays with the DNA samples extracted from blood culture bottles using Norgen isolation kit yielded 80% positivite result. However, the frequency of PCR positivite results was only 20% in the DNA samples extracted by Thermo DNA extraction kit. PCR result for GAPDH gene was also negative in 80% of the samples extracted by Thermo kit. Our results revealed that for the removal of inhibitors and detection of even low number of Brucella spp./B.melitensis in blood samples and blood culture bottles, NORGEN Blood DNA isolation kit can be used with a combination of QuantiTect multiplex PCR or Ampliqon Multiplex TEMPase.