Qu Yao, Liu Mengda, Sun Xiangxiang, Liu Yongxia, Liu Jianzhu, Hu Liping, Jiang Zhiqiang, Qi Fei, Nan Wenlong, Yan Xin, Sun Mingjun, Shao Weixing, Li Jiaqi, Sun Shufang, Zhang Haobo, Fan Xiaoxu
National Animal Tuberculosis Reference Laboratory, Division of Zoonoses Surveillance, China Animal Health and Epidemiology Center, Qingdao, Shandong, China.
College of Animal Technology, Shandong Agricultural University, Taian, Shandong, China.
Front Microbiol. 2024 Jun 14;15:1397792. doi: 10.3389/fmicb.2024.1397792. eCollection 2024.
Tuberculosis, caused by complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including , , and BCG, poses a potential challenge.
In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for , 4.47 copies/reaction for and 3.59 copies/reaction for BCG, without cross-reaction to , , , , , , , , and , and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 10 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of , , and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively.
Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of , , and BCG.
由结核分枝杆菌复合群(MTBC)引起的结核病,仍然是人类和动物的全球健康问题。然而,缺乏快速、准确和高度灵敏的检测方法来区分MTBC的主要病原体,包括结核分枝杆菌、牛分枝杆菌和卡介苗,这构成了一个潜在挑战。
在本研究中,我们建立了一种三重液滴数字聚合酶链反应(ddPCR)方法,采用三种类型的探针荧光团,其靶标分别为结核分枝杆菌(靶向RD1的CFP-10-ESAT-6基因和RD4的Rv0222基因)、牛分枝杆菌(靶向RD1的CFP-10-ESATs-6基因)和卡介苗(靶向ΔRD1的Rv3871和Rv3879c基因)。
基于退火温度、灵敏度和重复性的优化,该方法的检测下限(LOD)分别为结核分枝杆菌3.08拷贝/反应、牛分枝杆菌4.47拷贝/反应和卡介苗3.59拷贝/反应,对结核分枝杆菌、牛分枝杆菌、非洲分枝杆菌、田鼠分枝杆菌、堪萨斯分枝杆菌、海分枝杆菌、鸟分枝杆菌、胞内分枝杆菌、脓肿分枝杆菌和耻垢分枝杆菌无交叉反应,变异系数(CV)低于10%,显示出重复性。该方法对牛奶样品具有很强的耐受性,在加标牛奶中的检测限为5×10 CFU/mL,其灵敏度比三重qPCR高10倍。中国动物卫生与流行病学中心保存的60份临床DNA样品(包括20份牛奶、20份组织和20份拭子样品),采用三重ddPCR和三重qPCR进行检测。三重ddPCR的灵敏度(11.67%,7/60)高于三重qPCR方法(8.33%,5/60)。三重ddPCR检测结核分枝杆菌、牛分枝杆菌和卡介苗的阳性率分别为1.67%、10%和0%,三重qPCR分别为1.67%、6.67%和0%,符合率分别为100%、96.7%和100%。
我们的数据表明,建立的三重ddPCR方法是一种灵敏、特异且快速的区分和鉴定结核分枝杆菌、牛分枝杆菌和卡介苗的方法。