[Application of multiplex-PCR for bifidobacteria and propionibacteria genus identification]

Creation of a PCR test-system for determination of Actinobacteria class bacteria belonging to 2 genera that are the most widely represented among obligate anaerobic microbiota of human intestine: Bifidobacterium and Propionibacterium.

8 strains of Bifidobacterium spp. and 6 strains of Propionibacterium genus were identified by morphologic, cultural and biochemical properties. Isolation of matrix DNA of the strains for PCR was carried out by "DNA-Express" kit (SPF "Lytech", Russia). Primers for determination of genus membership for obligate anaerobes were developed based on variability of 16S RNA gene by using "Lasergene 7.1" ("DNASTAR, Inc.", USA) program. PCR screening of the isolated DNA was carried out based on "Syntol" LLC primers and reagents. Amplicon detection was carried out by agarose gel electrophoresis.

Multiplexing of 2 different primer pairs in a single probe at.68 degrees C annealing temperature for 30 cycles showed the presence of non-specific amplicons that form in samples with bifidobacteria DNA-matrix. The increase of annealing temperature to 70 degrees C and reduction of the number of PCR cycles to 25 resulted in the exclusion of formation of non-specific amplicons. Because the annealing temperature reached the level of values optimal for Taq-polymerase, a 2-phase PCR algorithm could be implemented. This solution allowed reducing the overall time of reaction to 45 minutes. Further increase of annealing temperature to 72 degrees C and reduction of elongation phase up to 15 seconds at 30 PCR cycles did not result in a visible reduction of reaction effectiveness:

A rapid system for identification of Bifidobacterium and Pronionibacterium genera using a system of 2-phase multiple PCR was developed. The system is part of a screening system for identification of major genera and species of cultured obligate anaerobic bacteria isolated from human intestine biotopes.