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Development and Application of a Multiplex qPCR Rapid Detection System for Syndromic Detection of Tick-Borne Pathogen
Bo Yi1,2,3, Ming-Qiu Fan3, Si-Yi Zhao4
1Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
Abstract:
Polymicrobial coinfections are increasingly becoming prevalent within Ixodidae tick vectors, which considerably complicates the global burden of tick-borne diseases. This reality necessitates rapid and accurate detection methodologies capable of concurrent pathogen detection. We engineered a multiplex quantitative polymerase chain reaction (qPCR) assay targeting Spotted Fever Group Rickettsiae (SFGR), Babesia microti, severe fever with thrombocytopenia syndrome virus, and Anaplasma phagocytophilum. Performance was evaluated on plasmid standard samples and 2050 field-collected ticks, benchmarking against established conventional PCR assay. The assay achieved 10 copies sensitivity, confirmed by probit regression (limit of detection95: 12.4-15 copies/µL) and robust quantification (R2 = 0.975-0.988, E = 95.630-101.668%). Receiver operating characteristic analysis suggested excellent preliminary accuracy (area under the curve: 1.000; sensitivity: 100.00%; specificity: 98.57-100.00% at a pre-specified Ct cutoff of 35). Clinical validation showed high concordance with conventional PCR for single pathogens (κ = 0.977-1.000; p < 0.001) and 100% agreement in quadruplex coinfection detection within the limited positive cases. Therefore, multiplex qPCR technology emerges as a sensitive, rapid, extremely specific, and efficient method for real-time syndromic surveillance of tick-borne polymicrobial infections, which enhances detection in regions burdened by overlapping vectors and interconnected zoonoses.
Insights
A new multiplex quantitative polymerase chain reaction (qPCR) assay can rapidly detect multiple tick-borne pathogens simultaneously. This technology enhances surveillance for complex coinfections in affected regions.
Area of Science:
- Veterinary Entomology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Polymicrobial coinfections transmitted by Ixodidae ticks are a growing global health concern.
- Current detection methods struggle with concurrent identification of multiple tick-borne pathogens.
- Accurate and rapid diagnostics are crucial for managing tick-borne disease burdens.
Purpose of the Study:
- To develop and validate a multiplex quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of key tick-borne pathogens.
- To evaluate the assay's performance against conventional methods using field-collected tick samples.
Main Methods:
- Engineered a multiplex qPCR assay targeting Spotted Fever Group Rickettsiae (SFGR), Babesia microti, severe fever with thrombocytopenia syndrome virus, and Anaplasma phagocytophilum.
- Assessed assay sensitivity, quantification, and accuracy using plasmid standards and 2050 field-collected ticks.
- Benchmarked performance against established conventional PCR assays.
Main Results:
- Achieved high sensitivity (10 copies) and robust quantification (R² = 0.975-0.988).
- Demonstrated excellent diagnostic accuracy (AUC = 1.000; sensitivity = 100.00%; specificity = 98.57-100.00%).
- Showed high concordance with conventional PCR for single pathogens and 100% agreement for quadruplex coinfections.
Conclusions:
- Multiplex qPCR is a sensitive, rapid, and highly specific tool for syndromic surveillance of tick-borne polymicrobial infections.
- This technology improves detection capabilities in regions with overlapping vectors and zoonotic diseases.
- Enables efficient real-time monitoring of complex tick-borne disease ecosystems.
