Establishment of LAMP detection system of Pyrenophora graminea.

[Objective] To provide a reference for detection of barley seeds and effective prevention and control of barley stripe, this study established a detection system for barley stripe based on loop-mediated isothermal amplification (LAMP) technology. [Method] In this study, five sets of specific primers were de-signed based on the ITS-rDNA sequences of P. graminea, and one set was screened to optimize reaction temperature based on six gradients (60, 61, 62, 63, 64 and 65 °C). Then, the LAMP system for efficient detection of the pathogen was established. Its specificity was tested based on LAMP amplification of DNA of genes from P. graminea, Ustilago hordei, Ustilago nuda, Dactylobotrys graminicola, Alternaria alternata and Fusarium graminearum. The sensitivity was analyzed by testing genes at levels of 310, 31, 3. 1, 0.31, 0.031 and 0.003 1 pg/µL. At last, it was used for testing and verifying seeds of five barley varieties. [Result] The established LAMP methods for detecting P. graminea performed optimally at 63 °C for 70 min with specific primers of set 2. Under the optimal reaction conditions, this method had good specificity for P. graminea with minimum detection limit of 0.31 pg/µL. The P. graminea-carrying rates of Dulihuang, Chaiqing 1, 1141, Beiqing 9 and Kunlun 15 seeds were 68.18%, 72.73%, 100%, 77.27% and 68.18%, respectively. [Conclusion] The established LAMP method can be used to detect the P. graminea in barley seeds within 70 min at 63 µC. [ABSTRACT FROM AUTHOR]