小熊猫紫色色杆菌PCR检测方法的建立及应用

Establishment and Application of PCR Method for Detection of Chromobacterium violaceum in Red Pandas

建立小熊猫紫色色杆菌PCR快速检测方法,根据紫色色杆菌毒力T3SS效应因子OspC家族蛋白的基因编码序列设计特异性引物,通过反应条件优化,引物特异性、灵敏性和重复性试验,建立PCR快速检测方法并应用于小熊猫病料和兽舍环境样品检测。结果表明,该PCR方法能特异性检测小熊猫紫色色杆菌,从小熊猫饲养环境和死亡小熊猫体内分离出其他菌株未扩增出条带;最低可检测出0.98 pg/μL的紫色色杆菌DNA,具有较好的重复性;用该检测方法从饲养环境和小熊猫病料中检测到的结果与传统的分离培养和测序比对的结果一致。所建立的PCR方法在实际应用中可快速高效检测出紫色色杆菌,可用于小熊猫紫色色杆菌病病原的检测。

To establish a rapid PCR assay for detection of Chromobacterium violaceum in red pandas,a pair of specific primers were designed according to the coding sequence of T3SS effector OspC family protein related to the virulence of Chromobacterium violaceum.Through the optimization of reaction conditions,primer specificity,sensitivity and repeatability tests,a rapid PCR detection method was established and applied to the detection of the diseased materials of red pandas and environmental samples of red panda enclosure.The PCR detection method could detect Chromobacterium violaceum of red pandas specifically,but showed negative to all the non-target strains isolated from the breeding environment and dead pandas.The detection sensitivity to Chromobacterium violaceum was 0.98 pg/μL,with good repeatability.The detection results of samples from feeding environment and diseased materials of red pandas were consistent with those of traditional isolation culture and sequencing.The established PCR detection method can be used to detect Chromobacterium violaceum rapidly and efficiently in practical applications,which can be used for the diagnosis of chromobacteriosis in red pandas and the detection of Chromobacterium violaceum.