Development of a Multiplex Primer Extension Assay for Rapid Detection of
            <i>Salmonella</i>
            Isolates of Diverse Serotypes

Ben-Darif, Elloulu; Jury, Francine; Pinna, Elizabeth de; Threlfall, E. J.; Bolton, F. J.; Upton, Mathew

doi:10.1128/jcm.01566-09

Cited by 23 publications

(12 citation statements)

References 32 publications

(26 reference statements)

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“…Numerous molecular techniques have been used to serologically type Salmonella isolates based on microarrays (5), real-time PCR (16), repetitive sequence-based PCR (25), multiplex primer extension (2), multilocus sequence typing (15), and bead-based arrays (6). Each of these methods has advantages and drawbacks in terms of cost, speed, robustness, and sensitivity (24).…”

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confidence: 99%

Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints

et al. 2012

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A classification model is presented for rapid identification of Salmonella serotypes based on pulsed-field gel electrophoresis (PFGE) fingerprints. The classification model was developed using random forest and support vector machine algorithms and was then applied to a database of 45,923 PFGE patterns, randomly selected from all submissions to CDC PulseNet from 2005 to 2010. The patterns selected included the top 20 most frequent serotypes and 12 less frequent serotypes from various sources. The prediction accuracies for the 32 serotypes ranged from 68.8% to 99.9%, with an overall accuracy of 96.0% for the random forest classification, and ranged from 67.8% to 100.0%, with an overall accuracy of 96.1% for the support vector machine classification. The prediction system improves reliability and accuracy and provides a new tool for early and fast screening and source tracking of outbreak isolates. It is especially useful to get serotype information before the conventional methods are done. Additionally, this system also works well for isolates that are serotyped as "unknown" by conventional methods, and it is useful for a laboratory where standard serotyping is not available.

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confidence: 99%

Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints

et al. 2012

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“…For small and medium-size laboratories, singleplex techniques became expensive and time-consuming when a large number of samples are evaluated. The alternatives to these methodologies, such as minisequencing assay – SNaPshot multiplex PCR –, have been successfully applied to support studies regarding oncogenic related genes [7-9], bacterial serotyping [10] and infectious diseases [11]. Moreover, the SNaPshot is a relatively simple and affordable method that allows more efficient and quick results when it is compared with singleplex techniques.…”

Section: Resultsmentioning

confidence: 99%

Three multiplex snapshot assays for SNP genotyping in candidate innate immune genes

et al. 2013

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BackgroundInnate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases. This immune response has been reported to be genetically diverse, due to polymorphisms coded by different genes. For this reason, our purpose was to develop a multiplex assay that allows the genotyping of candidate single nucleotide polymorphisms (SNPs) in innate immune genes.FindingsWe developed three multiplex PCR panels coupled with the minisequencing (SNaPshot) technique (multiplex PCR, multiplex primer extension, and capillary electrophoresis). The panels were tested in a sample set composed of 100 anonymous DNAs from healthy blood donors living in São Miguel Island (Azores, Portugal). Sixteen relevant SNPs among nine genes of the innate immune system – IL1α, IL1β, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14 – were genotyped and validated by direct sequencing, with the exception of one that was undetected by minisequencing. We suggest that these panels can be used in future studies for detection of risk gene variants in several populations and/or diseases.ConclusionsIn summary, we propose a multiplex assay that is able to identify the most frequent candidate SNPs in innate immune genes, using a medium scale genotyping platform. The assays can be used to evaluate the risk gene variants in populations of various geographic origins.

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“…The anxC4 gene has some discriminatory-informative single nucleotide polymorphisms (SNPs) that have been used in multilocus sequence typing (MLST) analysis (32). SNP markers have been applied for the detection and identification of Salmonella, Burkholderia cepacia, and A. fumigatus (32)(33)(34), so the variable anxC4 gene is suitable for use as a target for the detection and identification of A. fumigatus.…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

et al. 2016

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Aspergillus fumigatus is a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection of A. fumigatus infection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection of A. fumigatus. The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatus strains, including 5 species of the Aspergillus genus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10 2 copies for the same target. Clinical samples from a total of 69 patients with probable IA (n ‫؍‬ 14) and possible IA (n ‫؍‬ 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection of A. fumigatus in clinical testing has been developed.A spergillus fumigatus, a ubiquitous saprophytic fungus in most parts of the world, is a conditional pathogenic fungus and the major cause of invasive aspergillosis (IA). The infection is initiated by inhalation of conidia, which are cleared quickly in a normal host but can cause invasive disease in immunocompromised individuals (1). Despite many efforts during the last decade, the morbidity and mortality due to IA has been increasing (2). The poor therapeutic outcomes are associated with the weak host immune status and, importantly, the delayed detection and identification of the causative agent (3).The traditional culturing method, which is the gold standard for the diagnosis of A. fumigatus, requires several days for observation of growth, is time-consuming, and has only moderate specificity. Laboratory methods for the diagnosis of A. fumigatus include mainly direct microscopic examination, histopathological studies, and serum galactomannan (GM) and (1¡3)-␤-D-glucan assays. The measurement of galactomannan in bronchoalveolar lavage (BAL) fluid shows promise for enhancing the diagnosis of A. fumigatus (4-7), but only approximately 50% of BAL fluid measurement results can match the results of culture and direct examination (8).PCR-based molecular detection systems for fast and sensitive identification by examination of small amounts of DNA in serum and blood have been developed (9-11). However, the lack of standardization for critical activities in the methods and the evaluation of clinical samples has hampered the development of PCR-based diagnostic tests for clinical use....

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Development of a Multiplex Primer Extension Assay for Rapid Detection of Salmonella Isolates of Diverse Serotypes

Cited by 23 publications

References 32 publications

Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints

Prediction System for Rapid Identification of Salmonella Serotypes Based on Pulsed-Field Gel Electrophoresis Fingerprints

Three multiplex snapshot assays for SNP genotyping in candidate innate immune genes

Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus

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