Development of Species-specific Polymerase Chain Reaction (PCR) Targeting on Mitochondrial D-loop for Identification of Buffalo and Goat Raw Meat

Abstract: Today's, consumers are concerned about the meat they eat and also demandaccurate labeling. Mitochondrial Analysis of DNA was the most frequently used DNA, becauseof its highly conserved sequences in various organism species. In this study, a rapid,reproducible and simple method for simultaneous identification of multiple meat species ina single step mitochondrial DNA based test has been developed based on the designing ofspecies-specific primer. Meat samples of goat and buffalo were selected to verify theapplicability of the technique. A species specific forward and reverse primer was designedwith the help of the primer3 tool for amplification of mitochondrial D-loop region. Thespecies-specific primers were verified in silico by SnapGene software. The two pairs ofprimers amplified the expected fragment of 338bp for buffalo and 450bp for goat. The changein the size of the PCR product was due to the existence of highly polymorphic regions withinthe buffalo and goat D-loop region. The tested species gives a unique band pattern for eachspecies by using successful amplification of these polymorphic regions in the D-loop region.Overall, the simplicity of amplification of mitochondrial D-loop region could make thistechnique suitable for meat authentication in routine analysis.

Determination of Repeat Numbers of (CA)n in Mitochondrial D-loop using Polymerase Chain Reaction-single Strand Conformational Polymorphism (PCR-SSCP) / 체질인류학회지

Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis is a kind of sensitive mutation detection method that has been usually used in field of medical genetics. A single DNA strand with a mutation or nucleotide polymorphism has a different conformation from its wild-type counterpart, and these conformational differences result in different electrophoretic mobility. In previous study of mitochondrial microsatellite instability in 50 uterine leiomyomas, PCR-SSCP showed 4 types of band mobility at (CA)n of the mitochondrial D-loop. In type 1 and 4, positions of the lower single stand of both were same but those of upper strand were different. In sequencing analysis, repeat number of (CA)n in type 1 was 4, 5 in type 2, 6 in type 3, and 4 in type 4, respectively. Without using expensive sequencing analysis, PCR-SSCP method can be used to detect the repeat number of (CA)n in mitochondrial D-loop.

Investigation of the association between mitochondrial D-loop polymorphisms and hepatocellular carcinoma outcome / 中国比较医学杂志

Objective To investigate the accumulation of mutations and single nucleotide polymorphisms ( SNPs) in the displacement loop ( D-loop ) of mitochondrial DNA ( mtDNA ) might be associated with cancer risk and disease outcome.Methods We obtained cancerous and noncancerous liver tissues from 49 HBV-related HCC patients at the Fourth Hospital of Hebei Medical University.mtDNA of the liver tissues was extracted with Mitochondrial DNA Extraction Kit.Mutation and polymorphism were confirmed by repeated analysis.We assessed the prediction power of D-loop SNPs in hepatocellular carcinoma ( HCC) patients.Results No mutation in these HCC patients had prediction power for post-operational survival, whereas one SNP site ( nucleotide 150 C/T ) was identified by the log-rank test for statistically significant prediction of HCC survival.In an overall multivariate analysis, allele 150 was identified as an independent predictor of HCC outcome.The length of survival of patients with allele 150C was significantly shorter than that of patients with allele 150T (relative risk, 0.246;95% CI, 0.070–0.861; P=0.028).Conclusions The analysis of genetic polymorphisms in the mitochondrial D-loop helps to identify patient subgroups at high risk of a poor disease outcome.

Can Lutjanus purpureus (South red snapper) be "legally" considered a red snapper (Lutjanus campechanus)?

Red snappers (Lutjanus purpureus in Brazil and Lutjanus campechanus in USA and Gulf of Mexico) are both under clear effect of overfishing. Because of their high morphological similarity it has already been suggested that they could possibly be considered as a single species. To investigate the degree of similarity and the genetic structure of red snapper populations we constructed a common dataset of partial D-loop mtDNA sequences of L. purpureus from Brazil (Amapá, Pará and Maranhão) and L. campechanus from the Atlantic coast of the USA (Florida, Louisiana and Mississippi). Phylogenetic and population genetic analyses surprisingly depicted high similarity between L. campechanus and L. purpureus, compatible with the hypothesis of a single species of red snapper for the Western Atlantic Ocean. These preliminary but very curious findings open an important discussion regarding the legislation involved on the capture of this overexploited fish resources as well as regarding their taxonomy.