PCR−RFLP Analysis of Mitochondrial DNA:  A Reliable Method for Species Identification

Wolf, Christian; Rentsch, Jürg; Hübner, Philipp

doi:10.1021/jf9808426

Cited by 207 publications

(112 citation statements)

References 30 publications

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“…Confirmation of the meat species was according to Wolf et al [10]. Design of primers was based on the bovine Pth gene (K01938) and a 12S rRNA gene [8,21].…”

Section: Methodsmentioning

confidence: 99%

Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs

Chaumpluk

Chikae

Takamura

et al. 2006

Science and Technology of Advanced Materials

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Identifying contaminating bovine constituents in feed has been a major means to help prevent the spread of bovine spongiform encephalopathy (BSE). The phenomenon of DNA aggregation induced by Hoechst 33258 in conjunction with the change in anodic current measurement was, for the first time, applied for bovine DNA detection in feedstuffs. By using the PCR amplification system specific to bovine parathyroid and common 12S rRNA genes, anodic current peaks measurements of these PCR products on linear sweep voltammetry could be determined. Anodic peaks measurement of bovine parathyroid gene among ruminant meat and pet foods containing bovine constituents were at 1.18-1.52 mA while anodic peaks among non bovine samples were greater than 1.78 mA. In the study, anodic current peaks greater than 1.75 mA could be used to distinguish non-bovine from other samples in a qualitative analysis. For quantitative analysis, bovine content was measured using the comparative ratio between copy number of bovine parathyroid and 12S rRNA genes. This ratio reflected the proportion of target bovine cells to total cell numbers. In the experiment, contents of bovine constituents in four kinds of tested pet foods were 10.88, 8.76, 6.39 and 2.69%. Compared with the first two samples on which defined content had been addressed, the estimated content with 90.66 and 87.60% accuracy could be obtained, respectively. Although, this quantitative detection was not a real-time determination, the method had several merits on its rapidity and simplicity in performing the test and on its cost effectiveness since no sophisticated devices and expensive reagents were needed. q

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“…Confirmation of the meat species was according to Wolf et al [10]. Design of primers was based on the bovine Pth gene (K01938) and a 12S rRNA gene [8,21].…”

Section: Methodsmentioning

confidence: 99%

Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs

Chaumpluk

Chikae

Takamura

et al. 2006

Science and Technology of Advanced Materials

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“…PCR-RFLP analysis of a conserved fragment from the mitochondrial 12S rRNA has been applied to identify several closely related domestic animal and game species (Fajardo et al, 2006;Pfeiffer, Burger, & Brenig, 2004;Wolf, Rentsch, & Hubner, 1999).…”

Section: Introductionmentioning

confidence: 99%

“…However, the possible individual mutations make the selection of endonucleases in conserved restriction sites difficult, and several enzymes are often needed to discriminate between phylogenetically related species and when several species have to be differentiated simultaneously (Wolf et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis

Neve

Civera

Mucci³

et al. 2008

Meat Science

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The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species.The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.

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“…The restriction digestion pattern obtained from a combination of PCR amplification and restriction enzymes for RFLP analysis proved to be a suitable marker tool for the identification of these three species of cattle (Lenstra et al, 2001;AbdulMawjood and Bülte, 2002). Therefore, this study revealed the significant role that PCR-based techniques can play in species identification, and emphasizes the importance of choosing the right genes with suitable variation rates (Brown, 1983;Wolf et al, 1999). This method was successfully used to type both Malaysian wild and domestic cattle, and has previously been used in species identification of game and domestic meats (Prado et al, 2007;Fajardo et al, 2009;Wang et al, 2010;Chen et al, 2010).…”

Section: Discussionmentioning

confidence: 79%

Species identification of Malayan Gaur, Kedah-Kelantan and Bali cattle using polymerase chain reaction-restricted fragment length polymorphism

et al. 2014

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ABSTRACT. Mitochondrial DNA (mtDNA) is a useful genetic marker that can be used for species identification. The cytochrome b (Cyt b) gene is a suitable mtDNA candidate gene for use in phylogenetic analyses due to its sequence variability, which makes it appropriate for comparisons at the subspecies, species, and genus levels. This study was conducted to develop a rapid molecular method for species identification of Malayan gaur (Bos gaurus hubbacki), Kedah-Kelantan (KK) (Bos indicus), and Bali (Bos javanicus) cattle in Malaysia. DNA was extracted from blood samples of 8 Malayan gaurs, 30 KK, and 28 Bali cattle. A set of both specific and universal primers for the Cyt b gene were used in PCR amplification. DNA sequences obtained were then analyzed using BioEdit and Restriction Mapper softwares. The PCR products obtained from Cyt b gene amplification were then subjected to restriction enzyme Species identification of Malaysian cattle with PCR-RFLP digestion. The amplification, using both specific and universal primers, produced a 154-and a 603-bp fragment, respectively, in all three species. Two restriction enzymes, NlaIV and SspI, were used to obtain specific restriction profiles that allowed direct identification of Malayan gaur, KK, and Bali cattle. Our findings indicate that all three species can be identified separately using a combination of universal primers and the restriction enzyme SspI.

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PCR−RFLP Analysis of Mitochondrial DNA: A Reliable Method for Species Identification

Cited by 207 publications

References 30 publications

Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs

Novel electrochemical identification and semi quantification of bovine constituents in feedstuffs

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis

Species identification of Malayan Gaur, Kedah-Kelantan and Bali cattle using polymerase chain reaction-restricted fragment length polymorphism

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