Identification of shrimp species in raw and processed food products by means of a polymerase chain reaction-restriction fragment length polymorphism method targeted to cytochrome b mitochondrial sequences

A novel PCR-RFLP method has been developed for the identification of six commercially relevant penaeid shrimp species in raw and processed food products. The method can be completed within 8 h. To implement the method, PCR amplification with the crustF/crustR primers, targeted to the amplification of a ca. 181 bp region of the cytochrome b (cytb) mitochondrial gene in penaeid shrimps, was coupled to restriction analysis with CviJI, DdeI and NlaIV. The method was also applied successfully to the identification of shrimp species in complex processed foods, including this type of shellfish as an added-value food ingredient. The small size of this molecular target facilitates amplification from fresh, frozen, or precooked samples, where DNA fragmentation may be relevant and fragment size critical. We also report the first cytb mitochondrial sequences described to date for the species Farfantepenaeus notialis, Parapenaeus longirostris and Pleoticus muelleri, and these nearly triplicate current knowledge of reference nucleotide sequences in this mitochondrial region for this group of species. The cytb mitochondrial gene may also be considered as a molecular marker for identification and phylogenetic purposes in penaeid shrimp species.     

聚合酶链式反应-限制性片段长度多态性和芯片生物分析技术用于台湾海峡常见石斑鱼和鲷鱼的品种鉴定

应用聚合酶链式反应-限制性片段长度多态性分析(PCR-RFLP)和芯片生物分析系统建立了台湾海峡常见石斑鱼和鲷鱼的分子生物学品种鉴定新方法。首先提取鱼的基因组脱氧核糖核酸(DNA)进行细胞色素b基因特定片段的PCR扩增,然后用DdeI、HaeIII和NlaIII 3种限制性内切酶进行酶切,在Agilent DNA 1000芯片上对酶切片段进行分离。该方法成功鉴定了台湾海峡常见的8种石斑鱼品种和5种鲷鱼品种,是一种快速、简便、有效的鱼类品种鉴定分析手段。     

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

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.