Multiplex high resolution melt-curve real-time PCR assay for reliable detection of Salmonella

Food Control ◽  
2018 ◽  
Vol 91 ◽  
pp. 225-230 ◽  
Author(s):  
Yuejiao Liu ◽  
Prashant Singh ◽  
Azlin Mustapha
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Reliable Detection

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

2011 ◽  
Vol 64 (12) ◽  
pp. 2453-2459 ◽  
Author(s):  
G. N. van Blerk ◽  
L. Leibach ◽  
A. Mabunda ◽  
A. Chapman ◽  
D. Louw
Keyword(s):  
Surface Water ◽  
High Resolution ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Gene Target ◽  
Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

scholarly journals Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

2015 ◽  
Vol 59 (9) ◽  
pp. 5574-5580 ◽  
Author(s):  
Peera Hemarajata ◽  
Shangxin Yang ◽  
Janet A. Hindler ◽  
Romney M. Humphries
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Hydrolytic Activity ◽  
The United States ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Content Type ◽  
High Resolution Melt Analysis ◽  
Carbapenem Resistant

ABSTRACTThe rapid global spread of carbapenem-resistantEnterobacteriaceae(CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, usingblaOXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, includingblaKPC,blaSME,blaIMP,blaNDM-1,blaVIM,blaOXA-48,blaOXA-162,blaOXA-181,blaOXA-204,blaOXA-244,blaOXA-245, andblaOXA-232. Our assay identified the presence ofblaOXA-48-likeβ-lactamase genes and clearly distinguished betweenblaOXA-48and its variants in control strains, including betweenblaOXA-181andblaOXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.

scholarly journals Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3031-3040 ◽  
Author(s):  
Shabnam Rahimi-Khameneh ◽  
Sanni Hsieh ◽  
Renlin Xu ◽  
Tyler J. Avis ◽  
Sean Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phylogenetic Analyses ◽  
Similarity Index ◽  
Taqman Probe ◽  
Economic Losses ◽  
In Planta ◽  
Pcr Assay ◽  
Blind Test ◽  
Reliable Detection

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

scholarly journals Rapid Identification and Discrimination ofBrucellaIsolates by Use of Real-Time PCR and High-Resolution Melt Analysis

2010 ◽  
Vol 48 (3) ◽  
pp. 697-702 ◽  
Author(s):  
Jonas M. Winchell ◽  
Bernard J. Wolff ◽  
Rebekah Tiller ◽  
Michael D. Bowen ◽  
Alex R. Hoffmaster
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Identification ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis

Single-tube multiplex real-time PCR assay for rapid and reliable detection of HLA-A*31:01 allele

2018 ◽  
Vol 19 (10) ◽  
pp. 837-846 ◽  
Author(s):  
Tingting Zhang ◽  
Ying Xiao ◽  
Yanxia Wang ◽  
Yanwei Li ◽  
Lirong Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Single Tube ◽  
Reliable Detection

Development of multiplex real-time PCR and high-resolution melt analysis for detection of three viruses in penaeid shrimp

2010 ◽  
Vol 150 ◽  
pp. 127-127 ◽  
Author(s):  
Benjaporn Panichareon ◽  
Paisarn Khawsak ◽  
Warin Deesukon ◽  
Wasana Sukhumsirichart
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Penaeid Shrimp ◽  
High Resolution Melt ◽  
High Resolution Melt Analysis

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food

2016 ◽  
Vol 79 (5) ◽  
pp. 810-815 ◽  
Author(s):  
FEREIDOUN FORGHANI ◽  
SHUAI WEI ◽  
DEOG-HWAN OH
Keyword(s):  
Staphylococcus Aureus ◽  
Listeria Monocytogenes ◽  
High Resolution ◽  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Cost Savings ◽  
High Resolution Melt ◽  
And Staphylococcus Aureus

ABSTRACTThree important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 103 CFU/g without an enrichment step and 3.7 × 101 CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus.

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