DEVELOPMENT AND TESTING OF POLYMERASE CHAIN REACTION FOR INDICATION AND IDENTIFICATION OF ASPERGILLUS FLAVUS PHYTOPATHOGENIC FUNGI

Abstract

The article presents results of research on development of a test system by means of polymerase chain reaction with real-time detection for identification of Aspergillus flavus phytopathogenic fungi. The above microscopic fungi are contaminants of corn in various geographical areas. At present, a multiplex PCR-RT-test system "HRM-Zygo-Asp" is in operation for detection and identification of aspergillus (only to the genus) and a commercial test system for EIA "Aspergillus-IgG- EIA -BEST" (ZAO "VECTOR- BEST). The team of authors chose Aspergillus flavus strain CA14 4,044,380..4,045,732 b.p. for research, used Multiple Sequence Alignment Viewer 1.22.1 software, UGENA 44.0, NCBI BLAST-primer, Oligoevaluator. Specific primers were selected (f) 5'-3' GGGCCCGCAGCAAGAATAC, reverse primer - (r) 3'-5' ACGAGTTGTCACCTTCCCGAGA; a reaction protocol was developed, including preliminary denaturation - 95 0C - 5 minutes (1 cycle); denaturation - 95 0C - 5 sec, annealing - 60 0C - 15 sec (50 cycles). To determine the sensitivity of the test system, the authors selected a probe (CGGTTCGCTTTGGTCATCGT), a fluorescent dye - HEX, and a quencher - BHQ-2. It was determined that the sensitivity of the test system is 1000 cells. Approbation of the scientific development was carried out on 33 field strains isolated from 33 corn samples (grain, vegetative mass with signs of disease) and a reference strain (Aspergillus flavus VKM № F-25). When choosing field strains identified by V.I. Bilay and E.Z. Koval (1988) and the key of Nikitinskiy-Aleev, it was established that 25 samples were contaminated with bacteria of Aspergillus flavus species.