A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.
ABSTRACT The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. PRACTICAL APPLICATION: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.
- SourceAvailable from: José M Bautista[show abstract] [hide abstract]
ABSTRACT: The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNASer(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (OL) and the presence of a large polycytidine tract in the OL loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication.Journal of Molecular Evolution 01/1996; 41(6):942-51. · 2.15 Impact Factor
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ABSTRACT: By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.BioTechniques 10/1997; 23(3):504-11. · 2.40 Impact Factor