AI Article Synopsis
- Tuberculosis caused by Mycobacterium tuberculosis complex (MTBC) remains a major global health issue, emphasizing the need for effective detection methods to differentiate its main pathogens.
- The study introduces a new testing method called triplex droplet digital polymerase chain reaction (ddPCR), which uses different probe fluorophores to accurately target genes associated with three pathogens: MTBC, its variants, and BCG.
- The results show that triplex ddPCR is more sensitive and specific than current methods, detecting lower levels of pathogens in various sample types (including milk, tissue, and swabs) with high repeatability and without cross-reaction to other organisms.
Article Abstract
Introduction: 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.
Methods: 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.
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 , 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.
Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of , , and BCG.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11211260 | PMC |
| http://dx.doi.org/10.3389/fmicb.2024.1397792 | DOI Listing |
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