Haplotype-specific single-locus multiplex PCR assay for identification of Pacific and Atlantic white shrimps, exotic Litopenaeus vannamei and native Litopenaeus schmitti, along the coast of Brazil

This work describes a haplotype-specific single-locus multiplex PCR assay based on 16S mitochondrial gene polymorphisms for the identification of two Litopenaeus species present along the Brazilian coast: L. schmitti, native species from this genus in the area, and L. vannamei, exotic cultivated species that can be found along many sites along Brazil. Twenty-two 16S sequences of both species were used for the design of specific primers for each species. Two amplification patterns of the 16S gene are produced, one for each species, both with the amplification of two fragments: for L. schmitti, one universal 561 bp fragment and one specific 343 bp fragment are amplified; While for L. vannamei a universal 561 bp fragment and another species-specific fragment 408 bp is produced. For validation of the method we undertook a blind test where genetic profiles were analyzed by four different people with 100% accurate ratio. This system generates unequivocal genetic profiles to discriminate the two species of Litopenaeus in 2% agarose gels. The developed method is a useful identification tool for taxonomic purposes, in the control of the illegal trade of the native species in closed periods, as well as an instrument for monitoring the presence and spread of the exotic species in the natural environment coming from occasional escapes from shrimp farms. It provides an accurate system for direct identification of the two Litopenaeus species currently found on the Brazilian coast and also in other regions of the Atlantic where the two species co-exist.