Application of mitochondrial DNA analysis for microbial source tracking purposes in shellfish harvesting waters

2010 ◽  
Vol 61 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Craig Baker-Austin ◽  
Rachel Rangdale ◽  
James Lowther ◽  
David N. Lees
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Source Tracking ◽  
Pcr Analysis ◽  
Taqman Probes ◽  
Faecal Matter ◽  
Species Specific

We present a method for the reliable detection and source characterisation of faecal pollution in water and shellfish matrices, utilising real-time PCR analysis of mitochondrial DNA targets. In this study we designed real-time PCR (TaqMan) probes to target human, bovine, ovine and swine mtDNA. PCR amplification using species-specific TaqMan probes on faecal matter and mixed effluent slurries revealed no cross-reactions between species of interest and other vertebrate faecal matter. Performed as a single blind experiment we were able to correctly identify faecal material in 17/20 effluents (85% correct). mtDNA degrades relatively quickly in faecally-spiked water samples (∼2 weeks), a similar timeframe of environmental persistence to several bacterial faecal indictors, highlighting its applicability. The procedure described here is specific, rapid (<5 hours) and sensitive. These results confirm the suitability of using species-specific mtDNA as an indicator in source tracking studies in surface waters, shellfish harvesting areas and shellfish matrices.

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

scholarly journals Comparison between Various DNA Sterilization Procedures Applied in Forensic Analysis

2021 ◽  
Author(s):  
Nora Al Snan ◽  
Najib Alraimi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Uv Light ◽  
Dna Typing ◽  
Pcr Amplification ◽  
Forensic Analysis ◽  
Pcr Analysis ◽  
Dna Database ◽  
Template Dna ◽  
Study Laboratory

Abstract Background: The advanced sensitive STR kits applied in forensic DNA typing techniques can cause challenging issues when evidence samples are contaminated with minute quantities of DNA from another source such as forensic analysts or crime scene examiners. Methods and Results: In this study, laboratory air and surfaces, gloves, tools, and equipment were evaluated as potential sources of contaminating DNA. Different sterilization methods were tested for their ability to efficiently eliminate DNA in a sample. Inactivation methods included 10% bleach, ethanol, UV light and DNA-ExitusPlus IF. Exposure to the different inactivation protocols for varying periods of time was performed in two lab settings: low template DNA and DNA database labs. Surfaces were swabbed and any adhering DNA was quantified using HID real-time PCR. Results were detected using HID Real-Time PCR Analysis Software v1.2 and GeneMapper ID-X Software v1.4. Conclusions: It was concluded that most of the DNA decontamination methods are not suitable for highly sensitive and precision STR kits such as GlobalFiler PCR Amplification Kit. The most suitable tested method was using DNA-ExitusPlus IF with the incubation time increased from 10 to 15 minutes

scholarly journals Real-Time PCR Analysis of Vibrio vulnificus from Oysters

2003 ◽  
Vol 69 (12) ◽  
pp. 7137-7144 ◽  
Author(s):  
Mark S. Campbell ◽  
Anita C. Wright
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
Pcr Analysis ◽  
Viability Assay ◽  
Nonculturable Cells ◽  
Pure Cultures ◽  
Plate Counts ◽  
Opportunistic Human Pathogen ◽  
Species Specific

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (102 to 108 CFU ml−1), with a lower limit of 72 fg of genomic DNA μl of PCR mixture−1 or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r 2 = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.

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.

The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

2020 ◽  
Vol 21 (1) ◽  
pp. 225
Author(s):  
Abdul Rohman ◽  
Wiranti Sri Rahayu ◽  
Sudjadi Sudjadi ◽  
Sudibyo Martono
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Limit Of Detection ◽  
Specific Primer ◽  
Chain Reaction ◽  
Relative Standard ◽  
Polymerase Chain ◽  
Species Specific

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

Rapid quantification of Salmonella Typhimurium inoculated to meat products by real-time PCR

2009 ◽  
Vol 57 (1) ◽  
pp. 25-38 ◽  
Author(s):  
Ching-Yang Cheng ◽  
Jing-Ruei Chi ◽  
Sin-Rong Lin ◽  
Chi-Chiang Chou ◽  
Chin-Cheng Huang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat Products ◽  
Pcr Amplification ◽  
Food Samples ◽  
Pcr Analysis ◽  
Target Cells ◽  
Chicken Nuggets ◽  
Reliable Technique ◽  
Isolation And Purification

The objective of this study was to use a 5′-nuclease (TaqMan) real-time PCR method with primers and probe specific to thespaQgene as a rapid approach to quantitatively determineSalmonellaTyphimurium. The result showed that the correlation coefficient between real-time PCR estimates and bovine serum albumin (BSA) plate counts ofS. Typhimurium was 0.99, independently of 105-fold numbers of bystanderEscherichia coliO157:H7 or total viable counts. The sensitivity of the real-time quantitative PCR assay was 10 CFU/mL for pureS. Typhimurium culture without enrichment. A known number ofS. Typhimurium target cells were inoculated to dumpling fillings and chicken nuggets and DNA was extracted for real-time PCR analysis. The sensitivity was 60 CFU/g forS. Typhimurium inoculated to the food samples without any preceding procedure of enrichment. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h. This study demonstrated that realtime PCR is a rapid and reliable technique for quantifyingS. Typhimurium possessing thespaQgene in pure culture and in meat products.

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