Abstract: 【Objective】Morphological identification method is mainly used for detection and identification of Embellisia allii (Campanile) Simmons. The method is time-consuming in identification, moreover, the spore of garlic bulb canker is very similar to other species of the genus Embellisia and other genera, but is also very difficult to distinguish. Rapid and effective molecular diagnostic methods are needed. The objective of this study is to develop a real-time quantitative PCR (qPCR) technique for detection of the E. allii sensitively and accurately.【Method】From the NCBI GenBank database, 15 kinds of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene nucleic acid sequences of the garlic bulb canker and other similar species diseases were selected, according to primer design software Primer express, a probe and three pairs of primers were elected theoretically. Based on the experimental results of the above-mentioned primers, specific primers and TaqMan probes to E. allii were screened. While the reaction system renaturation-extension temperature of the reaction parameters were optimized, three temperature gradients of 56, 58, 60℃ were designed. The garlic bulb canker pathogen genomic DNA in the nucleic acid analyzer UV quantification was performed 10 times serial dilutions in order to test the probe sensitivity. The TaqMan qPCR detection system was established with optimized reaction conditions.【Result】The species-specific primer pair Emb21/Emb32 and probe Emb were designed based on gpd gene for E. allii, and E. allii could be detected specifically from the 6 tested strains with qPCR based on gpd gene, only the target fungi could be specifically detected positive, the blank control and other strains were detected negatively. About the optimization of refolding- extended temperature reaction parameters, △Rn fluorescence signal at 58℃ was the maximum while the value of the fluorescence signal was lower at 56, 60℃. After repeated tests, the optimal annealing-extended temperature was determined as 58℃. In the test of the sensitivity, the garlic bulb canker pathogen DNA concentration was diluted to 140 pg, resulting better graphics; when the system template concentration is diluted to 14 pg, the resulted graph was poor. The detection sensitivity of qPCR for DNA concentration was about 140 pg.【Conclusion】The qPCR detection system was established for E. allii. This detection technology can specifically detect E. allii from the 6 tested strains and the detection sensitivityis140 pg.The assay of qPCR based on gpd gene was specific and sensitive, and the detection method is suitable for E. allii rapid detection.

Key words: Embellisia allii, glyceraldehyde-3-phosphate dehydrogenase (gpd) gene, real-time quantitative PCR (qPCR)