Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR.

BACKGROUND: The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. METHODS: Five kits were evaluated using clinical specimens: QIAamp DNA Mini Kit (QIAGEN), peqGold Tissue DNA Mini Kit (PeqLab), UltraClean Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. RESULTS: Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared. CONCLUSIONS: We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

No correlation between giant cell arteritis and Chlamydia pneumoniae infection: investigation of 189 patients by standard and improved PCR methods.

A total of 189 temporal artery biopsy samples from giant cell arteritis (GCA) patients were investigated using sensitive PCR targeting Chlamydia pneumoniae. Chlamydial DNA was detected in 17 samples, 11 of which were positive for chlamydial antigens. Our data did not reveal strong evidence that C. pneumoniae plays an important role in the pathogenesis of GCA.

Ultra-rapid detection of Chlamydia trachomatis by real-time PCR in the LightCycler using SYBR green technology or 5'-nuclease probes.

Chlamydia trachomatis infections are among the most common sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid based detection method. Several commercial PCR test systems are available (e.g. CobasAmplicor, Roche, Mannheim, Germany) but they require post-amplification detection by hybridization resulting in extended work-up time and possible cross-contamination. The objective of our study was to develop a routine diagnostic method for the sensitive, specific and rapid detection of C. trachomatis. The obvious choice is real-time PCR without any post-amplification procedures. The dye SYBR Green I (intercalating in dsDNA) provides a simple and fast real-time PCR in the LightCycler. Specific primer design combined with melting curve analysis allows a reliable and sensitive identification of C. trachomatis. In addition, a new commercial real-time PCR system (RealArt C. trachomatis LC PCR Reagents, artus, Hamburg, Germany) was evaluated, that combines sequence-specific primers and fluorescence-labelled (FRET) 5'-nuclease probes. An internal control integrated in this system detects false negative results and erroneous PCR conditions. All results were compared with the corresponding data from an analysis using the CobasAmplicor system (Roche). (Clin

Comparison of diagnostic tests for the detection of Brucella spp. in camel sera.

BACKGROUND: Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of Brucella in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting Brucella infection in camels. FINDINGS: A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that bcsp31 kDa real-time PCR detected Brucella DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species. CONCLUSION: We suggest combining bcsp31 real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.

Preliminary validation of real-time PCR assays for the identification of Yersinia pestis.

BACKGROUND: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. METHODS: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. RESULTS: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. CONCLUSIONS: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.

Host cell responses to Chlamydia pneumoniae in gamma interferon-induced persistence overlap those of productive infection and are linked to genes involved in apoptosis, cell cycle, and metabolism.

The respiratory pathogen Chlamydia (Chlamydophila) pneumoniae is associated with chronic diseases, including atherosclerosis and giant-cell arteritis, which are accompanied by the occurrence of these obligate intracellular bacteria in blood vessels. There, C. pneumoniae seems to be present in a persistent state. Persistence is characterized by modified bacterial metabolism and morphology, as well as a reversible arrest of chlamydial development. In cell culture, this persistent state can be induced by gamma interferon (IFN-gamma). To elucidate this long-term interaction between chlamydiae and their host cells, microarray screening on epithelial HeLa cells was performed. Transcription of persistently (and productively) infected cells was compared with that of mock-infected cells. Sixty-six host cell genes were regulated at 24 h and/or 96 h of IFN-gamma-induced persistence. Subsequently, a set of 17 human host cell genes related to apoptosis, cell cycle, or metabolism was identified as permanently up- or down-regulated by real-time PCR. Some of these chlamydia-dependent host cell responses were diminished or even absent in the presence of rifampin. However, other expression patterns were not altered by the inhibition of bacterial RNA polymerase, suggesting two different modes of host cell activation. Thus, in the IFN-gamma model, the persisting bacteria cause long-lasting changes in the expression of genes coding for functionally important proteins. They might be potential drug targets for the treatment of persistent C. pneumoniae infections.

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples.

BACKGROUND: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. METHODS: We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. RESULTS: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. CONCLUSIONS: Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.