Rapid identification of maternal lineages in common carp (Cyprinus carpio L.) using real-time PCR and high resolution melt-curve analysis

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.

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OPTIMALISATION OF SPECIES IDENTIFICATION OF COMMON CARP (CYPRINUS CARPIO) USING SYBR® GREEN I REAL-TIME PCR METHOD

Potravinarstvo Slovak Journal of Food Sciences ◽

10.5219/154 ◽

2011 ◽

Vol 5 (3) ◽

Author(s):

Pavol Bajzík ◽

Radoslav Židek ◽

Jozef Golian ◽

Ľubomír Belej ◽

Jozef Čapla ◽

...

Keyword(s):

Real Time ◽

Species Identification ◽

Common Carp ◽

Cyprinus Carpio ◽

Real Time Pcr ◽

Common Carp Cyprinus Carpio ◽

Carp Cyprinus Carpio ◽

Pcr Method

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Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dku411 ◽

2014 ◽

Vol 70 (2) ◽

pp. 441-447 ◽

Cited By ~ 14

Author(s):

W.-S. Chan ◽

T.-M. Chan ◽

T.-W. Lai ◽

J. F.-W. Chan ◽

R. W.-M. Lai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Identification ◽

Curve Analysis ◽

Melt Curve Analysis ◽

Methicillin Resistant ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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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 ◽

10.1017/s0031182018001361 ◽

2018 ◽

Vol 145 (13) ◽

pp. 1733-1738 ◽

Cited By ~ 6

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.

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