Detection of Tilletia controversa with HRCA approach.
Objective: To establish a detection system of Tilletia controversa (TCK) with hyper-branched rolling cycle amplification (HRCA) method, which provides a stable, reliable and novel technique for early diagnosis of wheat dwarf bunt disease and identification of pathogen. Method: The padlock probe consists of a universal linking sequence and the two target complementary regions at 5′ and 3′ ends, which was designed based on the unique fragment sequence of 1 322 bp of TCK. Detection system of HRCA was established and optimized. The specificity and limitation of HRCA was determined and compared with conventional PCR. A total of 51 samples intercepted at Customs or collected from different wheat cultivation areas in USA and China were examined with HRCA. Result: HRCA is capable of amplifying mycelial and teliosporal DNA of TCK, while not detecting related smut species of Tilletia and other plant pathogen. The detection sensitivity of HRCA is as low as 1 fg.µl-1 for plasmid DNA and 10 pg.µl-1 for genomic DNA of TCK which is 10-fold higher than that of conventional PCR. The results show HRCA method to be sensitive, specific and accurate. Conclusion: The detection system of HRCA for TCK was successfully established, which provided a new approach for the simultaneous diagnosis of wheat dwarf bunt disease and identification of TCK pathogen.