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A Multiplex PCR Detection Assay for the Identification of Clinically Relevant Anaplasma Species in Field Blood
Yongshuai Peng1,2, Shanshan Zhao1, Kunlun Wang1
1College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.
Abstract:
The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10-3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10-5, 5 × 10-7, 2 × 10-5, and 7 × 10-7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.
Insights
A novel multiplex PCR assay enables the simultaneous detection of four Anaplasma species, improving diagnosis for sheep and goats. This sensitive and specific tool aids in rapid clinical diagnosis and epidemiological surveillance of Anaplasma infections.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- The genus Anaplasma causes significant infections in human and animal health globally.
- Multiple Anaplasma species infections are frequently reported, complicating diagnosis.
- Accurate and rapid diagnostic tools are crucial for Anaplasma management.
Purpose of the Study:
- To develop and validate a novel multiplex PCR assay for simultaneous detection of Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum.
- To assess the specificity, sensitivity, and reproducibility of the developed multiplex PCR.
- To provide an effective tool for differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in livestock.
Main Methods:
- Development of species-specific primers targeting groEL (A. capra, A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes.
- Evaluation of primer specificity using various blood pathogens.
- Sensitivity testing via 10-fold dilutions of infected sheep blood DNA.
- Reproducibility assessment using serial dilutions and field samples (n=175).
Main Results:
- The multiplex PCR demonstrated 100% species-specific detection.
- Lower limits of detection ranged from 5 × 10⁻⁷ to 3 × 10⁻⁵ ng/μL for the four Anaplasma species.
- No significant difference was observed between multiplex and simplex PCR protocols (P > 0.05).
- The groEL and 16S rRNA genes were confirmed as reliable targets for species identification.
Conclusions:
- The developed multiplex PCR is a sensitive, specific, and accurate method for simultaneous Anaplasma detection.
- This assay facilitates rapid differential diagnosis and epidemiological surveillance of key Anaplasma species in sheep and goats.
- The findings support the use of this tool for improved management of Anaplasma infections in veterinary settings.
