An SNP-Based Diagnostic Method for Brucella S2 Vaccine Strain Infections

Xingya Wang^1,2Xiaowei Tian^3,4Wanyang Li¹Yuanchao Yang¹Shuai Zhang¹Hui Wang¹Wanru Geng^3,4*Jingbo Zhai^1,5,6*

• ¹School of Basic Medical Sciences, Inner Mongolia Minzu University, Tongliao 028000, China
• ²Department of Laboratory, Hulunbuir Second People's Hospital, Hulunbuir 021000, China
• ³Department of Intensive Medicine, Affiliated Hospital of Inner Mongolia Minzu University, Tongliao, Inner Mongolia 028000, China
• ⁴College of Clinical Medicine, Inner Mongolia Minzu University, Tongliao, Inner Mongolia 028000, China
• ⁵Key Laboratory of Zoonose Prevention and Control at Universities of Inner Mongolia Autonomous Region, Tongliao 028000, China
• ⁶Brucellosis Prevention and Treatment Engineering Research Center of Inner Mongolia Autonomous Region, Tongliao 028000, China

Abstract: Background: Brucellosis, a zoonotic bacterial infection caused by Brucella species, exhibits a global distribution. The Brucella S2 vaccine strain is known to cause brucellosis. Current serological antibody assays cannot distinguish between infections caused by the S2 strain and those caused by wild-type Brucella. Objective: To develop a diagnostic method capable of specifically detecting S2 vaccine strain infections. Methods: Two probes were designed targeting single nucleotide polymorphism (SNP) loci upstream of the sugar ABC gene; quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) methods were established. The performances of these methods were evaluated. The transient stem-loop structure of the DNA template was predicted, and the impact of probe overlap with the transient stem-loop structure on detection sensitivity was analyzed. Clinical applicability was assessed using 50 blood samples from brucellosis patients. Results: Both types of methods demonstrated high specificity. However, MGB-SNPdd showed greater sensitivity than other detection methods. Reduction of overlap between the probe sequence and the transient stem-loop structure enhanced detection sensitivity. In the clinical applicability analysis, ddPCR methods exhibited higher rates of S2 vaccine strain detection compared with qPCR methods. Conclusion: SNP-based ddPCR methods demonstrate higher sensitivity than qPCR methods and enable specific detection of brucellosis caused by the S2 vaccine strain. Reduction of probe overlap with the transient stem-loop structure improves detection sensitivity, providing valuable insights for enhanced PCR amplification efficiency.