Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide

Abstract

Aims:The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. Methods and Results: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR Ò 2AE1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. Results and Conclusions: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. Significance and Impact of the Study: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.

Quantitative Real-Time PCR Analysis of FecalLactobacillusSpecies in Infants Receiving a Prebiotic Infant Formula

“…For the selection of primer and probe sequences, the 16S-23S intergenic spacer regions of the different Lactobacillus species were retrieved from the GenBank, EMBL, and DDBJ databases as follows: L. acidophilus (AB102855 [25], AF182726 [37], and U32971 [39]), L. alimentarius (AF500493 [33] and AF500492 [33]), L. amylovorus (AF182732 [37]), L. animalis (AY526616 and AY526614), L. brevis (AB102858 [25] and AF405353 [11]), L. bulgaricus (Z75475), L. casei (AB102854 [25], AF405352 [11], AF182729 [37], and AF121200 [38]), L. collinoides (AB117957 and AB117955), L. crispatus (AF182719 [37] and AF074857 [38]), L. curvatus (AF074858 [38], U97135 [5], and U97129 [5]), L. delbrueckii (]AB102856 [25], AB035485 [37], AB035484 [37], U32969 [39], U32968 [39], and U32967 [39]), L. farciminis (AF500491 [33] and AF500490 [33]), L. fermentum (AF182720 [37] [25], AF182721 [37], and AF074859 [38]), L. graminis (U97136 [5] and U97130 [5]), L. hamsteri (AF113601), L. helveticus (AF182728 [37]), L. jensenii (AB035486 [37] and U32970 [39] [37], AF182724 [37], and U32964 [39]), L. paralimentarius (AJ616014), L. paraplantarum (U97138 [5] and U97132 [5]), L. pentosus (U97141 [5], U97140 …”

Rapid Identification of Lactobacillus acidophilus by Restriction Analysis of the 16S–23S rRNA Intergenic Spacer Region and Flanking 23S rRNA Gene

“…It should be noted that the PCR products from each Lactobacillus strain always consisted of two ISR fragments, designated as small and large (Berthier & Ehrlich 1998, Song et al 2000, Rachman et al 2003. The nucleotide sequences of the major PCR products of the target size of approximately 650 bp were analyzed to determine which species in GenBank matched each strain with the highest homology.…”

“…Recently, variations in the lengths and sequences of rRNA intergenic spacer regions (ISRs) have been described; at the species level, the variation is greater among ISRs than among rRNA genes (Berthier & Ehrlich 1998, Song et al 2000, Tilsala-Timisjarvi & Alatossava 2001, Rachman et al 2003. Song et al (2000) used multiplex PCR assays with primers designed from nucleotide sequences of the 16S-23S rRNA ISRs and the flanking 23S rRNA genes for identification of lactobacilli from the human GI tract.…”

Leuconostoc mesenteroides subsp. suionicum subsp. nov.

“…Amplification of the 16S rRNA gene was performed using the primers of An et al (2006). The 16S-23S rRNA IGS was amplified using the primers and protocol of Rachman et al (2003). Purification and sequencing of PCR products were carried out by the Shenggong Company (Shanghai, China).…”