Test validation for the detection of Tilletia indica Mitra by multiplex real‐time PCR

Abstract

Tilletia indica Mitra is a fungal pathogen causing Karnal bunt of wheat. Tilletia indica is a quarantine pest in many countries worldwide. In the European Union, imported wheat grain from countries where the fungus is present must be checked for the presence of T. indica teliospores. The inspection services at the borders need rapid, sensitive and reliable detection tests to identify T. indica spores on wheat grain. In this work, validation was carried out according to EPPO Standard PM 7/98 to evaluate the multiplex real‐time PCR test described in ISPM 27 Diagnostic Protocol for regulated pests (Annex 4 Tilletia indica Mitra) by means of a test performance study with nine participating laboratories, and the performance characteristics of the test were established. The original protocol was modified with regard to the extraction of DNA from the pellet obtained from the ‘washing test’ and the enrichment PCR step in order to increase the amount of template DNA for the real‐time PCR. The optimized test still has five teliospores as the limit of detection for the contaminated pellet but has an increased analytical sensitivity and had positive results with three teliospores in 93% of cases instead of 43% for the original test. The two closest Tilletia species, Tilletia horrida and Tilletia walkeri, were used to evaluate analytical specificity (exclusivity) and no cross‐reactions were obtained. Diagnostic sensitivity, diagnostic specificity, accordance and concordance were also evaluated.