1. 1. Bacterial strains 35 Brucella culture isolates obtained from patients diagnosed with brucellosis were grown on chocolate agar media and the plates were incubated in a 5% CO 2 atmosphere at 37˚C for 48 hours. 2. DNA extraction DNA was extracted from pure cultures using the QIAamp DNA mini Kit (QIAGEN, Hilden, Germany). DNA concentrations and purity were measured by spectrophotometry. DNA dilutions were prepared and a total of 2 μl of the suspensions were used for PCR. 3. Real Time PCR Assays Real Time PCR was performed using the LightCycler  2.0 instrument (Roche Diagnostics, Mannheim, Germany). A- IS711 Assay: Primers and probes for the IS711 assay were designed after aligning all the IS711 sequences extracted from GenBank using Clustal_X (1.81) and they were as follows: Forward primer IS711-S [5’-TTGTCGATGCTATCGGCCTAC-3’] Reverse primer IS711-R [5’GGCAATGAAGGCCCTTA AGT-3’] Hybridization Probes IS711_FL [5’-GAAGCTTGCGGACAGTCACCATAAT-F-3’] and IS711_LC [5’-LCRed640-GCCGGGTGTTGGCTTTATTCG p-3’] (TIB MOLBIOL, GmbH Eresburgstraße-Berlin). Each reaction mixture contained 10 pmoles of each primer, 4 pmoles of each hybridization probe, 3mM MgCl 2, 2 μl of Fast Start Master Hybridization Probes reaction mix (Roche Diagnostics), and 2 μl of DNA extract, in a 20 μl total reaction volume. The PCR profile was as follows: 1) activation step of the Uracil-DNA Glycosylase at 40  C for 10 min; 2) activation of the Fast Start enzyme at 95  C for 10 min; 3) 45 cycles of template denaturation at 95°C for 10 s, primer annealing at 55°C for 10 s and primer extension at 72°C for 15 s, with a temperature transition rate of 20°C/s 4) Melting curve analysis: melting the template by heating to 95 °C then cooling to 45 °C for 30s then heating again to 95 °C with a temperature transition rate of 0.1°C/s. A tenfold dilution series was performed and samples containing 10 6 GE (genome equivalent) to 10 GE per reaction were run in duplicate to determine the standard curve. The log of the concentration was plotted versus the cycle number at which the fluorescent signal increased above threshold (Ct value). The slope of the standard curve generated allowed calculation of the reaction efficiency (E) according to the following equation: E=10 − (1/slope). Twofold dilutions were performed from 10 2 GEs down to ¼ GE and results from 8 replicates were used for probit regression analysis to establish the limit of detection (D95). To determine the specificity of the IS711 assay, 15 isolates of common gram- positive and gram-negative bacteria were tested. B. 16S-23S ITS Assay: The ITS assay developed by our group was performed using PCR conditions and cycling parameters similar to the IS711 assay. C. BCSP31 Hybridization Probe Assay: the TibMolBiol LightMix  Brucella genus detection kit assay was performed as recommended by the manufacturer. D. BCSP31 SYBR Green I Assay: this assay was performed as previously described (Queipo-Ortuno MI et al, Clin Microbiol Infect 11: 713-718, 2005). 4. Statistical analysis Calibration curves (Log GE versus Ct values) for the 4 assays were plotted in Microsoft Excel. Probit regression analysis was performed in SPSS version 10.0. Pairwise comparisons of probit curves for the four assays were performed using the bprobit function in STATA version 6, as follows: bprobit [number of positives][total number of replicates][number of GE per reaction][assay format as a binary 0/1 variable]. The D95 and D50 values for each assay were inferred from probit curves in SPSS. MATERIALS & METHODS Development of a Real-Time PCR Assay with Hybridization Probes for Sensitive Detection of Brucella Species Mireille M. Kattar, Simon Khalife, Roland J. Abi Rached, George F. Araj Department of Pathology and Laboratory Medicine American University of Beirut Beirut - LEBANON C-274  IS711 is an insertion sequence specific for the genus Brucella. Seven copies of IS711 are present in B. melitensis and B. suis, and 6 or 7 copies are present in B. abortus.  A real-time PCR assay targeting IS711 is more sensitive than assays targeting loci with a lower copy number in Brucella genomes including the 16S-23S ITS locus and bcsp31. Both targets are highly conserved in Brucellae. This real-time PCR assay could become the gold standard in molecular diagnosis of brucellosis.  The ITS assay targeting a locus present in 3 identical copies in Brucella genomes is more sensitive than bcsp31 assays. Bcsp31, present at 1 copy in Brucella genomes, is the most widely used PCR target in diagnostic assays for human and animal brucellosis.  No difference was observed between SYBR Green I and hybridization probe assays targeting bcsp31, suggesting that assay sensitivity on the LightCycler platform is not affected by the detection format but rather by target abundance. Hybridization probe assays are usually more specific than sequence- independent SYBR Green I assays.  Assay sensitivity determined by probit analysis was not affected by the variation in PCR efficiency. All assays had an efficiency > 1.80.  A linear correlation was observed between D50 (concentration where 50% of replicates are positive) and the number of target copies per genome equivalent. These data suggest that whenever possible, more abundant targets should be used in molecular diagnostic assays. DISCUSSION & CONCLUSIONS  Brucellosis is a widespread zoonosis, which affects a variety of livestock and wildlife, resulting in a decreased reproductive efficiency and abortion. Human infections occur in many developing countries. In humans, brucellosis is a highly diverse illness which initially presents as a febrile nonspecific syndrome and may later develop into a chronic disease affecting various organs and tissues. Hence, the diagnosis is frequently delayed or even missed.  The causative agents of brucellosis are facultative intracellular gram-negative bacteria belonging to the genus Brucella.  According to cultural, metabolic, antigenic properties and host specificity, eight species have been recognized, of which four have been associated with clinical disease in humans. These include B. melitensis that usually infects sheep and goats, B. abortus in cattle, B. suis in swine, and B. canis in dogs.  The laboratory diagnosis of brucellosis has relied on culture and serological methods. Brucella species grow slowly in culture media and are biochemically inert, thus requiring 1-2 weeks or longer for isolation and definitive identification. Serological assays suffer from lack of standardization and may be difficult to interpret in endemic regions.  Several studies have shown that the sensitivity of conventional PCR may be similar or even superior to culture.  It has been demonstrated that real-time quantitative PCR (QPCR) may be more sensitive than conventional PCR. QPCR is also faster, less laborious and minimizes the risk of carry-over amplicon contamination.  Recently described real-time PCR assays with SYBR Green I, TaqMan, and hybridization probes have shown variable sensitivity in detecting Brucella species DNA directly in clinical samples.  We have shown that patients with brucellosis have a low-level bacteremia thus requiring development of sensitive assays for detection. Real-time PCR coupled with the choice of a PCR target at multiple copies per genome-equivalent (GE) is likely to improve the lower limit of detection (LOD) of the assay.  In this study, we have developed a real-time PCR assay with hybridization probes targeting the highly conserved brucella- specific insertion sequence IS711; which is present at 6-7 copies per genome-equivalent in Brucella species. This assay was compared to three other previously described real-time PCR assays: 1) the 16S-23S internal transcribed spacer (ITS) region present at 3 copies per GE; 2) the TibMolBiol LightMix  Brucella genus detection kit, a HybProb assay targeting bcsp31 coding for the 31 KDa cell surface protein (present at 1 copy/GE) 3) a newly described SYBR Green I assay, also targeting bcsp31 (Queipo-Ortuno et al, 2005). INTRODUCTION Background: Human and animal brucellosis are widely endemic worldwide. The laboratory diagnosis of brucellosis by culture and serology has been enhanced by conventional PCR assays. Patients with brucellosis have a low-level bacteremia thus requiring development of rapid and sensitive assays for detection. Real-time PCR combined with the choice of a PCR target at multiple copies per genome equivalent (GE) is likely to improve the lower limit of detection (LOD). Methods: A hybridization probe LightCycler  assay (HybProb) was developed targeting the brucella-specific insertion sequence IS711, present at 6-7 copies per GE in Brucella species. The linear range, efficiency and LOD of this assay determined by probit regression analysis, were compared to those of three other brucella-specific assays: 1) a previously developed HybProb assay targeting the 16S-23S internal transcribed spacer region (ITS) (present at 3 copies/GE); 2) the TibMolBiol LightMix  Brucella genus detection kit, a HybProb targeting bcsp31 (present at 1 copy/GE); 3) a newly described SYBR Green I assay, also targeting bcsp31. Results: The IS711 detected 35 consecutive clinical Brucella species isolates and showed 100% specificity when tested on 15 isolates of common gram-positive and gram-negative bacteria. The respective LODs and efficiencies of the 4 assays were as follows: 1- IS711: 2.35 GE and 1.94; 2- ITS: 12.08 GE and 1.98; 3- bcsp31 HybProb: 53.12 GE and 1.82; 4- bcsp31 SYBR Green I: 55.1 GE and 1.87. The linear range for all assays was 10 2 to 10 6 GE. IS711 was more sensitive than ITS (p< 0.001), and ITS was more sensitive than both bscp31 HybProb and SYBR Green I (p=0.002 and p=0.022, respectively). The difference in sensitivity between both bcsp31 assays was not statistically significant (p=0.9). The D50 (GE copies where 50% of replicates are positive) was inversely correlated with the target copy number (C) per GE (D50= 29.559/C – 4.5273; R 2 = 0.99). Conclusion: The IS711 assay is more sensitive than ITS, and bcsp31 for detection of Brucella species. This assay could constitute a useful adjunct for the laboratory diagnosis of brucellosis. ABSTRACT RESULTS