An integrated strategy of multiplex qPCR and BclA gene length polymorphism analysis for differentiating Bacillus...

A combined molecular strategy was developed to accurately differentiate wild-type and vaccine strains of Bacillus anthracis. This approach integrates a multiplex TaqMan qPCR assay targeting the BclA, sap, and cap genes with conventional PCR analysis of BclA fragment length polymorphism. The qPCR assay demonstrated high specificity, with no cross-reactivity against other common pathogens, and exhibited a consistent limit of detection of 2.11 × 10¹ copies/μL (Cq < 35) for all three targets. Excellent repeatability was confirmed, with intra- and inter-assay coefficients of variation below 1.96% and 3.06%, respectively. Validation using 19 wild-type strains revealed two distinct qPCR profiles, and all wild-type strains were conclusively differentiated from the capsule-producing Chinese vaccine strain II and the acapsular Sterne strain by their characteristic BclA amplicon sizes. Through simulation PCR validation of common global canSNP genotyping (groups A, B, and C), it is possible to distinguish between wild-type and vaccine strains in China. This integrated "multiplex qPCR-BclA length analysis" protocol provides a reliable, sensitive, and specific tool for the precise detection and differentiation of B. anthracis strains in China.IMPORTANCEAnthrax, which is caused by the bacterium B. anthracis, remains a serious zoonotic disease. Its effective surveillance relies on precise strain identification. Currently, it is challenging to rapidly and accurately distinguish wild-type strains from vaccine strains, which hinders outbreak investigation and response. This study develops a novel molecular strategy combining multiple genetic targets to solve this critical diagnostic problem. The new method provides a reliable tool for strain differentiation, directly supporting accurate source tracing and informed decision-making during anthrax outbreaks. By improving diagnostic precision, this work contributes to strengthening public and veterinary health defenses against this persistent threat.