Определение аналитической чувствительности и специфичности методов ПЦР для диагностики черной бактериальной пятнистости томата

Abstract

The work is devoted to molecular genetic methods for the diagnosis of pathogens of bacterial spot of tomato – a harmful bacterial disease of tomatoes and peppers, predominantly spread in the open ground in the southern regions. The research was conducted on the basis of the All- Russian Plant Quarantine Center (FGBU “VNIIKR”) in 2022–2023. The objective of the work was to determine the analytical sensitivity and specificity of molecular genetic methods for the detection and identification of three species of different pathological types of bacteria of the genus Xanthomonas – pathogens of bacterial spot of tomato. Due to the necessity of import substitution of expensive reagents, the tests were carried out with domestic reagents produced by OOO “Evrogen” and ZAO “Dialat”. The results of the effectiveness of real-time PCR (qPCR) are presented, the analysis of which showed that all tested primer systems can be recommended for the detection of bacteriosis pathogens. At the same time, qPCR AFLP derived Taqman PCR and XopD Taqman PCR were found to have high analytical sensitivity (102–103 CFU/ml). It has been shown that three out of four primer systems according to to the methodology of Koenraadt et al. (2009) also have a high sensitivity of 102 CFU/ml. The analytical sensitivity of the Bs-XpF/R primers for the detection of X. euvesicatoria pv. perforans was 104 CFU/ml, which is sufficient for samples with a high concentration of the pathogen and for the identification of a pure bacterial culture. All primers are 100% specific for the target strains of pathogens; no cross-reactions with other strains have been observed. The proposed reliable operational method for detecting pathogens of bacterial spot in tomato and pepper seeds will significantly reduce yield losses and increase the economic efficiency of domestic vegetable production.