致病性与非致病性溶血性曼氏杆菌双重TaqMan荧光定量PCR鉴别检测方法的建立

Development of duplex TaqMan real-time PCR assay for differential detection of pathogenic and nonpathogenic Mannheimia haemolytica

为建立一种快速鉴别检测致病性与非致病性溶血性曼氏杆菌的双重TaqMan荧光定量PCR方法,根据溶血性曼氏杆菌16S rRNA和白细胞毒素(lktA)基因保守序列设计特异性引物及探针,并优化反应条件,建立了鉴别致病性与非致病性溶血性曼氏杆菌的双重TaqMan荧光定量PCR检测方法。结果显示:所建立的双重TaqMan荧光定量PCR方法与其他24种常见牛羊细菌均无交叉反应,具有较好的特异性;其检测lktA基因阳性和阴性溶血性曼氏杆菌的最低检出限度均为40 copies,具有较好的敏感性。应用建立的双重荧光定量PCR方法和普通PCR方法对48份临床样品进行检测,这2种方法检出的溶血性曼氏杆菌阳性率分别为27.1%和18.8%,检出lktA基因阳性溶血性曼氏杆菌的阳性率分别为20.8%和12.5%,阳性符合率为100%。本研究中建立的双重荧光定量PCR方法为牛羊溶血性曼氏杆菌致病性与非致病性菌株感染的高通量快速鉴别诊断提供了可行手段。

To establish a rapid differential detection method of pathogenic and nonpathogenic Mannheimia haemolytica(Mh),a duplex Taq Man real-time PCR assay was developed with two pairs of specific primers and two Taq Man probes designed according to the conserved region of the 16 S rRNA and leukotoxin gene lktA of Mh and the reaction conditions were optimized.The results showed that this method was specific for detecting Mh and had no cross-amplifications for other 24 bacteria species of cattle and sheep and had good specificity.The assay could differentially detect lktA gene positive and lktA gene negative Mh with the detection limit 40 copies of the genomic DNA and had higher sensitivity.The assay and conventional PCR were used to simultaneously detect 48 clinical samples of cattle,sheep and goat,the results showed that the Mh positive rates were 27.1% and 18.8%,respectively,and the lktA gene positive Mh positive rates were 20.8 % and 12.5 %, respectively, with positive coincidence rate was 100%.The established real-time PCR assay provided a credible way for rapid and high through-put differential diagnosis of the infection of pathogenic and nonpathogenic Mh in cattle,sheep and goat.