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. 2024 Jun 14:15:1397792.
doi: 10.3389/fmicb.2024.1397792. eCollection 2024.

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG

Yao Qu #  1   2 , Mengda Liu #  1   3 , Xiangxiang Sun  1   3   4 , Yongxia Liu  2 , Jianzhu Liu  2 , Liping Hu  5 , Zhiqiang Jiang  1 , Fei Qi  1 , Wenlong Nan  1   3 , Xin Yan  1   4 , Mingjun Sun  1 , Weixing Shao  1 , Jiaqi Li  1 , Shufang Sun  1   3 , Haobo Zhang  1   3 , Xiaoxu Fan  1   3
Affiliations

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG

Yao Qu et al. Front Microbiol. .

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Discussion: 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.

Keywords: BCG; M. bovis; M. tuberculosis; molecular diagnosis; multiplex droplet digital PCR; tuberculosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
ddPCR assay for primers and probes screening. (A–C) 3 sets primers and probes screening for RD1. (D–F) 3 sets primers and probes screening for RD4. (G–I) 3 sets primers and probes screening for ΔRD1.
FIGURE 2
FIGURE 2
Determination of the optimal probe concentrations of the ddPCR of RD1 (A), RD4 (B), ΔRD1 (C).
FIGURE 3
FIGURE 3
Screening the optimum annealing temperature from 55 to 62°C of RD1 (A), RD4 (B), ΔRD1 (C).
FIGURE 4
FIGURE 4
Performances of the triplex ddPCR assay by target DNA from 3 × 104 to 3 × 100 copies/μL. (A–C) Detection results of the target M. tuberculosis. (D–F) Detection results of the target M. bovis. (G–I) Detection results of the target BCG.
FIGURE 5
FIGURE 5
Standard curve of M. tuberculosis, M.bovis and BCG. (A,B) show the standard curves of triplex ddPCR and triplex qPCR, respectively, and (C) indicates the correlation between them.
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