RESUMEN
Identification of members of the Lactobacillus sakei group (LSG) by common phenotypic and genotypic methods is generally inadequate and time-consuming. The objective of this study was to develop novel species-specific primers based on sequence-characterized amplified region (SCAR) markers using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Three species-specific fragments were gel-purified, cloned and sequenced after preliminary screening of 80 random primers. Accordingly, three pairs of primers Lcur-F/R, Lgram-F/R and Lsakei-F/R were designed based on single species-specific bands (281, 278 and 472 bp) that were obtained from Lactobacillus curvatus, Lactobacillus graminis and L. sakei, respectively. The specificities of these primer pairs were confirmed in 21 LSG strains and 31 nontarget Lactobacillus strains. In addition, the detection limits for each primer pair were approx. 105 , 104 and 106 cells per gram of meat samples spiked with L. curvatus, L. graminis and L. sakei, respectively. In conclusion, we have successfully developed a rapid, accurate and effective PCR-based method for identification of species in the LSG. SIGNIFICANCE AND IMPACT OF THE STUDY: Neither phenotypic nor the most commonly used genotypic method (16S rRNA gene sequencing) provides sufficient resolution for accurate identification of the Lactobacillus sakei group. A sequence-characterized amplified region method developed in this study provides a rapid, cost-effective way to detect the member of the L. sakei group in meat sample.
Asunto(s)
Latilactobacillus sakei/clasificación , Latilactobacillus sakei/genética , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Técnica del ADN Polimorfo Amplificado Aleatorio/métodos , Cartilla de ADN/genética , ADN Bacteriano/genética , Marcadores Genéticos/genética , Genotipo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Background Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.