A polymerase chain reaction‐restriction fragment length polymorphism method based on the analysis of a 16S rRNA/tRNA^Val mitochondrial region for species identification of commercial penaeid shrimps (Crustacea: Decapoda: Penaeoidea) of food interest

Abstract

A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species of food interest belonging to the superfamily Penaeoidea. Preliminary amplification, sequencing and alignment of a 960 bp fragment of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial region allowed the design of 16Scru4/16Scru4 primers, constructed on well-conserved mitochondrial sequences of the penaeid shrimp species considered. Such primers afforded the amplification of an internal 515-535 bp region of the 16S rRNA/tRNA(Val) genes that, when subjected to cleavage with AluI, TaqI and HinfI, provided species-specific restriction patterns. Moreover, the proposed method also allowed the definition of different intraspecific restriction types between different populations of Litopenaeus vannamei, Farfantepenaeus notialis, Fenneropenaeus merguiensis, Metapenaeus sp., Melicertus latisulcatus and Pleoticus muelleri of different origins. The method described here was also successfully applied for the identification of penaeid shrimps in complex processed precooked foods, where this type of shellfish is used as an added-value food ingredient. Sequencing analysis provided new information about the genetic relationships among shrimps not only at the levels of species and genus, but also among different populations at intraspecific level. The 16S rRNA/tRNA(Val) fragment considered in this study seems to be accurate for shrimp species identification in raw and processed foodstuffs and for phylogenetic analysis among penaeid shrimp species.

Species identification through mitochondrial rRNA genetic analysis

“…The mitochondrial 12S rRNA and/or 16S rRNA genes have been used as molecular markers to identify mammals, birds, shrimp, and other species using species-specific primers that amplify the 12S rRNA or 16S rRNA gene regions from mtDNA2324252627282930313233343536373839. Universal mitochondrial 12S rRNA gene primers can amplify corresponding regions from a wide variety of organisms, including birds and insects26.…”

“…The mitochondrial 12S rRNA gene was used in an earlier study to differentiate snail species according to a PCR-RFLP approach26. Other researchers have used species-specific mitochondrial 12S rRNA and/or 16S rRNA gene primers to identify species of shrimp, fish, poultry, fruit flies, and snakes (from venom), among others 2728293031323334353637383940…”

Mitogenomic variation of Bangladesh Penaeus monodon (Decapoda, Panaeidae) and reassessment of its phylogeography in the Indo-West Pacific region

“…Variation in the mitochondrial DNA has been widely assessed for taxonomic identification and in phylogeographic and population genetic studies e.g. control region (CR) (Walther et al, 2011;Vaseeharan et al, 2013), and the 16S rRNA gene and a downstream fragment of the 16S rRNA and tRNA Val genes (Lavery et al, 2004;Kumar et al, 2007;Pascoal et al, 2008;Vaseeharan et al, 2013). The highest mtDNA diversity in P. monodon was found in Indonesia, with less diversity identified in south-east Africa and west Australia (Benzie et al, 2002).…”

Identification of European species of <i>Maja</i> (Decapoda: Brachyura: Majidae): RFLP analyses of COI mtDNA and morphological considerations

“…This species can only be confused with juveniles of the two larger European species of Maja. Identification of crustacean species using PCR-RFLP analysis has been well documented (Power et al, 1999;Bossier et al, 2004;Khamnamtong et al, 2005;Hisar et al, 2008;Pascoal et al, 2008;Dharani et al, 2009). The PCR-RFLP technique, to be diagnostic, must be applied to DNA regions that are highly conserved within species but sufficiently variable between species.…”