A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

Abstract

Aims: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy‐to‐perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S‐23S rDNA intergenic spacer region (ISR). Methods and Results: PCR amplification of the 16S‐23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro‐organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L‐ISR), containing tRNAIle and tRNAAla genes; small spacer (S‐ISR) without tRNA genes. The third described spacer (medium, M‐ISR), original for L. sanfranciscensis, harboured a tRNA‐like structure. An oligonucleotide sequence targeting the variable region between tDNAIle and tDNAAla of L. sanfranciscensis L‐ISR was approved to be suitable in species‐specific identification procedure. Analysis by pulse‐field gel electrophoresis of the chromosomal digest with the enzyme I‐CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1·3 Mb. Conclusions: Direct amplification of 16S‐23S ISRs or PCR with specific primer derived from L‐ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. Significance and Impact of the Study: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.

Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses

“…Bacterial DNA was extracted using an Archive Pure DNA Purification System (5 PRIME) according to the manufacturer's instructions. A preliminary identification was performed through the recognition of the electrophoretic profile generated from the amplification of internal ribosomal spacers according to Valcheva et al (2007). Furthermore, all the strains were subjected to the sequencing of a portion of the 16S rRNA-coding gene in order to confirm that they belonged to the species.…”

“…Through the elaboration of results obtained from plate counts the generation time was calculated to be 4 h. A few colonies at the highest dilutions were randomly picked at each monitoring time. Species identification was carried out by the analysis of the internal ribosomal spacers, as described by Valcheva et al (2007).…”

“…In fact, the possibility of extracting amplifiable microbial DNA directly from panettone cake after heat treatment, and consequently of identifying the species involved in the fermentative process, has recently been demonstrated (Picozzi et al, 2006). Genetic characterization at the strain level has already been evaluated using different techniques (Zapparoli et al, 1998;Randazzo et al, 2005;Catzeddu et al, 2006;Corsetti et al, 2007;De Angelis et al, 2007;Valcheva et al, 2007). Although random amplified polymorphic DNA (RAPD)-PCR assays have been presented as a suitable tool to detect genetic polymorphism within a species, it is often difficult to compare the results from different studies, since they are heavily dependent on the experimental operating conditions.…”

A competitive quantitative polymerase chain reaction method for characterizing the population dynamics during kimchi fermentation

“…Therefore, the conventional method using selective media is not ideal for monitoring fermentative processes from a quantitative aspect; it takes a relatively long period of time to get results and only the organisms that have the ability to survive in an artificial medium can be enumerated. As molecular biology techniques have been developed, quantitative PCR methods for detecting 16S rRNA gene sequences [2,4] or the 16S-23S intergenic spacer regions [20] have been developed using specific PCR primers. However, since these primers are often sensitive to little changes in annealing temperature, multiple gene amplification may occur and it can prevent accurate identification of microbes.…”

Bio-Diversity of Lactobacillus Cultures Associated with the Traditional Ethnic Fermented Foods of West Garo Hills, Meghalaya, India

“…Adeymo et al, (2014) similarly reported the characterisation of Lactobacillus plantarum using molecular methods by polymerase chain reaction (PCR) and amplification of 16S rDNA genes to confirm their identities from fermented cereals. Direct amplification of 16S-23S intergenic space regions (ISRs) or PCR with specific primer derived from L-ISR was reported to be useful for specific typing of Lactobacillus sanfranciscensis (Valcheva et al, 2006).…”