Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment.
Ontology highlight
ABSTRACT:
SUBMITTER: McConnell MJ
PROVIDER: S-EPMC3318517 | biostudies-literature | 2012 Apr
REPOSITORIES: biostudies-literature
Publications
Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment.
McConnell Michael J MJ Pérez-Ordóñez Ana A Pérez-Romero Pilar P Valencia Raquel R Lepe José Antonio JA Vázquez-Barba Isabel I Pachón Jerónimo J
Journal of clinical microbiology 20120201 4
A real-time PCR assay was developed for detecting the presence of Acinetobacter baumannii on hospital equipment and compared to conventional bacterial culture using 100 hospital environmental samples. The real-time PCR detected contaminated surfaces in 4 h with high sensitivity (100%) compared to conventional culture. Thirty-eight percent of samples were positive by real-time PCR and negative by bacterial culture (false positives), possibly indicating the widespread presence of bacterial DNA tha ...[more]
Similar Datasets
Project description:Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.
| S-EPMC93119 | biostudies-literature
Project description:Acinetobacter baumannii is a Gram-negative bacterium considered an emerging multi-drug-resistant pathogen. Furthermore, this bacterium can survive in extreme environmental conditions, which makes it a frequent cause of nosocomial infection outbreaks. Gene expression analyses by Reverse Transcription Quantitative real-time PCR (RT-qPCR) depend on a reference gene, also called an endogenous gene, which is used to normalize the generated data and thus ensure an accurate analysis with minimal errors. Currently, gene expression analyses in A. baumannii are compromised, as there are no reports in the literature describing the identification of validated reference genes for use in RT-qPCR analyses. For this reason, we selected twelve candidate reference genes of A. baumannii and assessed their expression profile under different experimental and culture conditions. The expression stability of the candidate genes was evaluated by using statistical algorithms such as BestKeeper, geNorm, NormFinder, Delta CT, and RefFinder, in order to identify the most suitable candidate reference genes for RT-qPCR analyses. The statistical analyses indicated rpoB, rpoD, and fabD genes as the most adequate to ensure accurate normalization of RT-qPCR data in A. baumannii. The accuracy of the proposed reference genes was validated by using them to normalize the expression of the ompA gene, encoding the outer membrane protein A, in A. baumannii sensible and resistant to the antibiotic polymyxin. The present work provides suitable reference genes for precise RT-qPCR data normalization on future gene expression studies with A. baumannii.
| S-EPMC10869792 | biostudies-literature
Project description:BackgroundDetection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.Methodology and significant findingsSpecies-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.ConclusionThe RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.
| S-EPMC3699609 | biostudies-literature
Project description:Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect B. burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of B. burgdorferi was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the B. burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of B. burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.
| S-EPMC84995 | biostudies-literature
Project description:Human bocavirus (HBoV) was discovered in 2005 and is associated with respiratory tract symptoms in young children. Three additional members of the genus Bocavirus, HBoV2, -3, and -4, were discovered recently from fecal specimens, and early results indicate an association between HBoV2 and gastrointestinal disease. In this study, we present an undifferentiating multiplex real-time quantitative PCR assay for the detection of these novel viruses. Differentiation of the individual bocavirus species can be subsequently achieved with corresponding singleplex PCRs or by sequencing. Both multiplex and singleplex assays were consistently able to detect ≤10 copies of HBoV1 to -4 plasmid templates/reaction, with dynamic quantification ranges of 8 logs and 97% to 102% average reaction efficiencies. These new assays were used to screen stool samples from 250 Finnish patients (median age, 40 years) that had been sent for diagnosis of gastrointestinal infection. Four patients (1.6%; median age, 1.1 years) were reproducibly positive for HBoV2, and one patient (0.4%; 18 years of age) was reproducibly positive for HBoV3. The viral DNA loads varied from <10(3) to 10(9) copies/ml of stool extract. None of the stool samples harbored HBoV1 or HBoV4. The highly conserved sequence of the hydrolysis probe used in this assay may provide a flexible future platform for the quantification of additional, hitherto-unknown human bocaviruses that might later be discovered. Our results support earlier findings that HBoV2 is a relatively common pathogen in the stools of diarrheic young children, yet does not often occur in the stools of adults.
| S-EPMC3020864 | biostudies-literature
Project description:Adenovirus infection is becoming increasingly recognized as a cause of morbidity and mortality in the immunosuppressed patient population. While early detection and quantitation of adenovirus in peripheral blood has been suggested as a means of directing and monitoring antiviral therapy in these patients, few methods have been published, particularly with respect to viral quantitation. A multiplexed real-time PCR assay was developed that can quantitatively detect a wide range of known serotypes of human adenovirus, including all of subgroups A to C. This assay was compared to a qualitative, Southern blot-based PCR assay by using 45 peripheral blood specimens from 16 patients. There was 100% concordance between the two tests in terms of qualitative results. The real-time assay detected adenovirus in patient samples at levels from <200 to 266,681 copies/ml of blood. By using control viral samples, sensitivity was demonstrated to less than 10 copies of viral genome per reaction and quantitative linearity was demonstrated from 10 to 10(6) copies of input viral DNA. Equivalent sensitivity and linearity were demonstrated for 15 different reference serotypes of adenovirus. Eleven other viral serotypes have complete target region sequence homology to one or more of the strains tested. No cross-reactivity was noted with other commonly isolated viral species. Sequence analysis showed no significant homology with any other human pathogens (bacterial or viral). This assay allows rapid, sensitive, and specific quantitation of adenovirus and may have a significant impact on the care of immunocompromised patients at risk for disseminated viral infection.
| S-EPMC254346 | biostudies-literature
Project description:A real-time PCR assay was developed for the detection of Ehrlichia chaffeensis. The assay is species specific and provides quantitative results in the range 10 to 10(10) gene copies. The assay is not inhibited by the presence of tick, human, or mouse DNA and is compatible with high sample throughput. The assay was compared with previously described assays for E. chaffeensis.
| S-EPMC179802 | biostudies-literature
Project description:Detection of species in nature at very low abundance requires innovative methods. Conventional PCR (cPCR) and real-time quantitative PCR (qPCR) are two widely used approaches employed in environmental DNA (eDNA) detection, though lack of a comprehensive comparison of them impedes method selection. Here we test detection capacity and false negative rate of both approaches using samples with different expected complexities. We compared cPCR and qPCR to detect invasive, biofouling golden mussels (Limnoperna fortunei), in samples from laboratory aquaria and irrigation channels where this mussel was known to occur in central China. Where applicable, the limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate of each PCR method were tested. Quantitative PCR achieved a lower LoD than cPCR (1 × 10-7 vs. 10-6 ng/μl) and had a higher detection rate for both laboratory (100% vs. 87.9%) and field (68.6% vs. 47.1%) samples. Field water samples could only be quantified at a higher concentration than laboratory aquaria and total genomic DNA, indicating inhibition with environmental samples. The false negative rate was inversely related to the number of sample replicates. Target eDNA concentration was negatively related to distance from sampling sites to the water (and animal) source. Detection capacity difference between cPCR and qPCR for genomic DNA and laboratory aquaria can be translated to field water samples, and the latter should be prioritized in rare species detection. Field environmental samples may involve more complexities-such as inhibitors-than laboratory aquaria samples, requiring more target DNA. Extensive sampling is critical in field applications using either approach to reduce false negatives.
| S-EPMC6303721 | biostudies-literature
Project description:A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.
| S-EPMC87012 | biostudies-literature
Project description:The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.
| S-EPMC1636138 | biostudies-literature