Correction: Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.
Ontology highlight
ABSTRACT:
SUBMITTER: Verstappen KM
PROVIDER: S-EPMC5718433 | biostudies-literature | 2017
REPOSITORIES: biostudies-literature
Publications
Correction: Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.
Verstappen Koen M KM Huijbregts Loes L Spaninks Mirlin M Wagenaar Jaap A JA Fluit Ad C AC Duim Birgitta B
PloS one 20171206 12
[This corrects the article DOI: 10.1371/journal.pone.0183925.]. ...[more]
Similar Datasets
Project description:Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.
| S-EPMC5578505 | biostudies-literature
Project description:Here, we report the complete and draft genome sequences of 8 Staphylococcus pseudintermedius isolates, 4 from human bacteremia infections and 4 from canine bacteremia infections. This species is recognized primarily as an important canine pathogen, but it is increasingly being identified in human infections.
| S-EPMC6624775 | biostudies-literature
Project description:A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.
| S-EPMC120553 | biostudies-literature
Project description:BACKGROUND: WGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples. RESULTS: Aliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance. CONCLUSION: We investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 x 10(-4)). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results.
| S-EPMC2675535 | biostudies-literature
Project description:Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C T ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4) colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
| S-EPMC3583986 | biostudies-other
Project description:BackgroundMolecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation.ResultsIn silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay.ConclusionsThis assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.
| S-EPMC8188689 | biostudies-literature
Project description:Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log(10) versus 4 log(10) copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R(2) ≥ 0.98 in each of 6 regression models) and clinical samples (R(2) = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.
| S-EPMC3553899 | biostudies-literature
Project description:We report the first complete genome sequences of three predominant clones (ST68, ST71, and ST84) of methicillin-resistant Staphylococcus pseudintermedius in North America. All strains were isolated from canine infections and have different SCCmec elements and antibiotic resistance gene patterns.
| S-EPMC5073276 | biostudies-literature
Project description:Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.
| S-EPMC193789 | biostudies-literature
Project description:BackgroundsPCR is a proper technique that significantly improves toxoplasmosis diagnosis. However, a more sensitive technique is required. This study compared real-time PCR with nested PCR using B1, SAG-4, and MAG-1 bradyzoite genes to diagnose toxoplasmosis in toxoplasmic retinochoroiditis patients.MethodsBlood samples were collected from 10 patients with active toxoplasmic chorioretinal lesions and 10 healthy individuals. Blood samples including peripheral blood mononuclear cells (PBMCs), serum and whole blood samples were used for DNA extraction. Serum was also used to detect anti-toxoplasma IgG and IgM antibodies. Nested PCR and real-time PCR were performed using B1, SAG-4, and MAG-1 target genes.ResultsFive (50%) out of the 10 patients were tested positive for toxoplasmosis with nested PCR using the PBMC samples. All the five patients tested positive with nested PCR were also tested positive for toxoplasmosis with real-time PCR using the PBMC samples. The real-time PCR results demonstrated that 9(90%) out of the 10 patients were positive based on B1 and the remaining one (10%) was positive only based on MAG-1. In general, of the patients, five (50%) were positive using SAG-4 and three (30%) were positive in term of MAG-1 using PBMCs with real-time PCR.ConclusionIt appears that PBMC samples have the best performance as the PCR extraction method and are a good source for toxoplasmosis diagnosis. The use of B22 and B23 target genes due to their high sensitivity and specificity along with bradyzoite genes are recommended for toxoplasmosis diagnosis using PBMC samples with real-time PCR.
| S-EPMC8609853 | biostudies-literature