APPROVAL OF THE TEST-SYSTEM FOR THE INDICATION AND IDENTIFICATION OF ASPERGILLUS FLAVUS BY THE METHOD OF POLYMERASE CHAIN REACTION IN THE “REAL TIME” MODE

Abstract

the article presents the results of studies on approbation of a test system for the indication and identification of microscopic fungi Aspergillus flavus by the polymerase chain reaction method with real-time detection. Using software Multiple Sequence Alignment Viewer 1.22.1 and UGENA 44.0. The test system for A. flavus includes specific primers: forward primer (f) 5’-3’ GGGCCCGCAGCAAGAATAC, reverse primer (r) 3’-5’ ACGAGTTGTCACCTTCCCGAGA; fluorescent dye: HEX, probe - CGGTTCGCTTTGGTCATCGT, quencher BHQ2. Reaction protocol: preliminary denaturation - 95 0C - 5 minutes (1 cycle); denaturation - 95 0C - 5 sec, annealing - 60 0C - 15 sec (50 cycles). Probe: AGCATAGGCTGATGCTCGTAGGC, fluorescent dye - ROX, quencher - BHQ-2. The sensitivity of the test system is 1000 cells. The optimal concentration of primers was set equal to 9 pM of each primer per reaction. The optimal probe concentration is 0.4 pM. The results obtained indicate that the use of dichotomous keys does not allow the most accurate differentiation of phytopathogenic fungi A. flavus. A new approach to the identification of isolates confirmed the belonging of 15 isolated strains to the species A. flavus out of 20 isolated from corn samples with signs that manifest themselves as root rot and wilting, and initially typed as Aspergillus based on the study of cultural and morphological properties. The study was carried out according to the thematic plan-task of the Ministry of agriculture of the Russian Federation, the registration number of the INIS RTD 122030200367-8.