PDF(Pubmed)
Abstract：
UNASSIGNED: Tuberculosis, caused by Mycobacterium tuberculosis 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 M. tuberculosis, M. bovis, and BCG, poses a potential challenge.
UNASSIGNED: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
UNASSIGNED: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, 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 × 103 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 M. tuberculosis, M. bovis, 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.
UNASSIGNED: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.
UNASSIGNED: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
UNASSIGNED: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, 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 × 103 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 M. tuberculosis, M. bovis, 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.
UNASSIGNED: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.
摘要：
■结核病，由结核分枝杆菌复合体（MTBC）引起，仍然是人类和动物的全球健康问题。然而,缺乏快速，准确,和高灵敏度的检测方法来区分MTBC的主要病原体，包括结核分枝杆菌,M.Bovis,和BCG,构成了潜在的挑战。
■在这项研究中，我们建立了使用三种类型的探针荧光团的三重液滴数字聚合酶链反应（ddPCR）方法，与目标结核分枝杆菌（靶向CFP-10-ESAT-6基因的RD1和RD4的Rv0222基因），牛分枝杆菌（靶向RD1的CFP-10-ESATs-6基因），和BCG（靶向ΔRD1的Rv3871和Rv3879c基因），分别。
■基于退火温度的优化，灵敏度和重复性，该方法证明了结核分枝杆菌的检测下限（LOD）为3.08拷贝/反应，牛分枝杆菌4.47拷贝/反应，卡介苗3.59拷贝/反应，对溶血曼海姆菌没有交叉反应，牛支原体,副猪嗜血杆菌,大肠杆菌,多杀性巴氏杆菌,嗜血杆菌,霍乱沙门氏菌,布鲁氏菌，和金黄色葡萄球菌,并显示出重复性，变异系数（CV）低于10％。该方法具有很强的牛奶样品耐受性，在穗乳中检测的LOD为5×103CFU/mL，其灵敏度是三重qPCR的十倍。60份临床DNA样本,包括20份牛奶,20个组织和20个拭子样本，分别保存在中国动物卫生与流行病学中心，通过三重ddPCR和三重qPCR进行检测。三重ddPCR呈现更高的灵敏度(11.67%，7/60）比三重qPCR方法（8.33％，5/60）。结核分枝杆菌的阳性率，M.Bovis,通过三重dddPCR，卡介苗分别为1.67、10和0%，和1.67，6.67，和0%的三重qPCR，符合率为100％，96.7％和100％，分别。
■我们的数据表明，建立的三重ddPCR方法是一种灵敏的，用于鉴别和鉴定结核分枝杆菌的特异性和快速方法，M.Bovis,和BCG。
■在这项研究中，我们建立了使用三种类型的探针荧光团的三重液滴数字聚合酶链反应（ddPCR）方法，与目标结核分枝杆菌（靶向CFP-10-ESAT-6基因的RD1和RD4的Rv0222基因），牛分枝杆菌（靶向RD1的CFP-10-ESATs-6基因），和BCG（靶向ΔRD1的Rv3871和Rv3879c基因），分别。
■基于退火温度的优化，灵敏度和重复性，该方法证明了结核分枝杆菌的检测下限（LOD）为3.08拷贝/反应，牛分枝杆菌4.47拷贝/反应，卡介苗3.59拷贝/反应，对溶血曼海姆菌没有交叉反应，牛支原体,副猪嗜血杆菌,大肠杆菌,多杀性巴氏杆菌,嗜血杆菌,霍乱沙门氏菌,布鲁氏菌，和金黄色葡萄球菌,并显示出重复性，变异系数（CV）低于10％。该方法具有很强的牛奶样品耐受性，在穗乳中检测的LOD为5×103CFU/mL，其灵敏度是三重qPCR的十倍。60份临床DNA样本,包括20份牛奶,20个组织和20个拭子样本，分别保存在中国动物卫生与流行病学中心，通过三重ddPCR和三重qPCR进行检测。三重ddPCR呈现更高的灵敏度(11.67%，7/60）比三重qPCR方法（8.33％，5/60）。结核分枝杆菌的阳性率，M.Bovis,通过三重dddPCR，卡介苗分别为1.67、10和0%，和1.67，6.67，和0%的三重qPCR，符合率为100％，96.7％和100％，分别。
■我们的数据表明，建立的三重ddPCR方法是一种灵敏的，用于鉴别和鉴定结核分枝杆菌的特异性和快速方法，M.Bovis,和BCG。
