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 Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species

Parasitology ◽  
2018 ◽  
Vol 145 (13) ◽  
pp. 1733-1738 ◽  
Author(s):  
Lucas J. Cunningham ◽  
J. Russell Stothard ◽  
Mike Osei-Atweneboana ◽  
Samuel Armoo ◽  
Jaco J. Verweij ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
Strongyloides Stercoralis ◽  
Curve Analysis ◽  
Pcr Analysis ◽  
High Resolution Melt ◽  
Middle Income ◽  
Ancylostoma Duodenale ◽  
Species Specific

AbstractWith the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato–Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

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

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S140-S140
Author(s):  
A Kalam
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Income ◽  
Water Samples ◽  
Real Time Pcr ◽  
Tap Water ◽  
Watery Diarrhea ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

2012 ◽  
Vol 95 (6) ◽  
pp. 1652-1655 ◽  
Author(s):  
Rakesh Kumar ◽  
K V Lalitha
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Gene Probe ◽  
Pcr Assay ◽  
Nylon Membrane ◽  
Rapid Enumeration ◽  
Salmonella Serovars ◽  
Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

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