THE ISOLATION AND IDENTIFICATION OF LACTIC ACID BACTERIA USING THE RFLP-PCR METHOD OF THE GENE 16 rRNA
І.M. Slyvka1, O.Y. Tsisaryk1, T. Bocer2
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1Lviv National University of Veterinary Medicine and Biotechnologies named
after S. Z. Gzhytskyj, Pekarska st., 50, Lviv, 79010, Ukraine
2University of Rzeszow, Sokolowska str., 26, Kolbuszowa, 36-100, Poland
The application of modern molecular genetic methods for identification of lactic acid bacteria (LAB) is one of the criteria for the selection strains of LAB wich are perspective for use in biotechnology and the food industry. Our purpose was to identify LAB, isolated from ewe’s cheese that is traditionally produce in the Carpathian region of Ukraine. We used a method of analysis of restriction fragment length polymorphism DNA (RFLP) based on polymerase chain reaction (PCR).We used a method of analysis of restriction fragment length polymorphism DNA (RFLP -PCR). 28 pure cultures of LAB were isolated from prototype cheese of bacteria of the genus Lactobacillus. 12 reference strains bacteria of the genus Lactobacillus were used for a comparative analysis of DNA restriction fragment.
The isolation of genomic DNA from cultures was carried out with using a set of Genomic Mini (A&A Biotechnology). Two primers EGE1 and 1492R (Sigma-Aldrich) used for amplification of the 16S rRNA gene. Two enzymes RsaI and HinfI used for dissection amplified DNA.
Several different restriction fragment DNA profiles made it possible to identify method RFLP-PCR.
Three groups of bacteria using the enzyme HinfI and four groups of bacteria using enzyme RsaI were identified similarity location restriction fragment DNA investigating strains of LAB in agarose gel. Two types of bacteria of the genus Lactobacillus identified based on comparative analysis of DNA restriction fragment investigating strains of LAB reference strains. Species Leuconostoc mesenteroides ssp. mesenteroides identified when using enzyme RsaI and species Lactococcus lactis ssp. identified when using the enzyme HinfI.
The appropriate is to determine the affinity of selected of LAB detection of heterogeneous properties and partial definition of belonging to the species investigating strains of bacteria using the RFLP-PCR method.
Keywords: LACTIC ACID BACTERIA, GENOTYPING, POLYMERASE CHAIN REACTION, AMPLIFICATION, POLYMORPHISM
