Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Comparative clinical study of different multiplex real time PCR strategies for the simultaneous differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis

Background: Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis. Methodology/Principal Findings: We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%). Conclusions/Significance: In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.