Development Of Lamp And Real-time Pcr For Detection Of Acidovorax Citrulli And Xanthomonas Oryzae

Acidovorax citrulli, Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv. oryzae were declared as the significant quarantine disease by Chinese govemment.They were typical seedborne pathogen. At present, A. citrulli has become great threat to melon industry, while, Xanthomonas oryzae has led to negative impact on food production. For this reason, ensuring the exclusion of infested seeds by applying the effective, commercially viable and convenient detecting technologies is crucial for the food industry.In the present study, Real-time PCR and LAMP assay were developed for detection of A.citrulli, Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv. oryzae, and specific primers and probe were designed based on the target genes, respectively. We detected the specificity, sensitivity and applicability of pure cultures and seed samples. Meanwhile, comparative evaluation was done between Real-time PCR and LAMP assay. The results showed that Real-time PCR and LAMP assay could distinguish Acidovorax avenae, Acidovorax facilis, Acidovorax konjaci and Acidovorax cattleyae with A.citrulli in the detection of A.citrulli, and seed pathogen carrier rate was0.1%(1/1000). In addition, using the LAMP and Real-time fluorescent PCR assay,6of13melon seedlots collected from Xinjang province were positive for A. citrulli. Porbe xooc-Probe and xoo-Probe could specifically detect all strains of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, Separately, the detection cycle was2h. LAMP technology realized the same specific amplification, and the detection time was1h. In detection of health rice seeds and seeds collected from disease areas, two methods for Xanthomonas oryzae pv. oryzicola could detected2of2commercial seed pathogen,1of2natural seeds, and0of7of disease areas seeds inoculated leaf blight pathogen were positive for Xanthomonas oryzae pv. oryzicola; The two detection methods of Xanthomonas oryzae pv. oryzae could detecte0of2commercial seed pathogen,1of2natural seeds,4of7of disease areas seeds inoculated leaf blight pathogen were positive for Xanthomonas oryzae pv. oryzae. The detection sensitivity of the three pathogen was up to2.0×101cps/μL for Taqman Real-time PCR, while the sensitivity of LAMP was up to2.0×102cps/μL. Apparently, the sensitivity of Taqman Real-time PCR is10copies higher than LAMP. In the case of same specificity and sensitivity, LAMP was potentially useful for diagnosing infection of the three pathoge in field amplication when took testing cycle, cost and applicability in account.