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Microchip electrophoretic separation for the fast diagnosis of Anaplasma phagocytophilum infection in cattle
Doori Oh1, Mi-Jin Lee, Jinho Park
1Department of Chemistry and Research Institute of Physics and Chemistry, Chonbuk National University, Jeonju, Republic of Korea.
Abstract:
We report a diagnostic method for Anaplasma phagocytophilum (A. phagocytophilum) infection in cattle using a nested PCR and microchip electrophoresis (ME). A. phagocytophilum causes human granulocytic anaplasmosis and granulocytic ehrlichiosis, which are emerging tick-borne zoonotic diseases. Nested PCR was used to amplify genomic DNA samples extracted from cattle blood. The amplified PCR products were analyzed under a sieving gel matrix of 0.7% poly(ethyleneoxide) (M(r)=8,000,000) in a conventional glass microchip. In the ME assay, A. phagocytophilum was analyzed within 35 s with a relative standard deviation of 1.30% (n=5) using a programmed field strength gradient (PFSG) as follows: 615.3 V/cm for 0-24 s, 66.7 V/cm for 24-34 s, 615.3 V/cm for 34-100 s. The ME-PFSG assay was clinically validated by comparing the 16S rRNA gene levels obtained by this method with those measured using conventional slab gel electrophoresis performed with ten cattle blood samples suspected of A. phagocytophilum infection. In contrast to slab gel electrophoresis, the proposed ME-PFSG methodology had increased sensitivity (200-450 pg/microL), a faster analysis time (<35 s), and required a smaller sample volume (approximately 162 fL).
Insights
A new diagnostic method using nested PCR and microchip electrophoresis (ME) accurately detects Anaplasma phagocytophilum in cattle. This faster, more sensitive assay offers improved detection of this emerging tick-borne zoonotic disease.
Area of Science:
- Veterinary Diagnostics
- Molecular Biology
- Biotechnology
Background:
- Anaplasma phagocytophilum is an emerging tick-borne zoonotic pathogen.
- Cattle are susceptible to A. phagocytophilum, causing significant economic impact.
- Accurate and rapid diagnostics are crucial for disease control.
Purpose of the Study:
- To develop and validate a novel diagnostic method for Anaplasma phagocytophilum infection in cattle.
- To improve upon existing diagnostic techniques in terms of speed, sensitivity, and sample volume.
- To utilize nested PCR combined with microchip electrophoresis (ME) for enhanced detection.
Main Methods:
- Genomic DNA was extracted from cattle blood samples.
- Nested PCR was employed to amplify the 16S rRNA gene of A. phagocytophilum.
- Amplified products were analyzed using microchip electrophoresis (ME) with a programmed field strength gradient (PFSG).
Main Results:
- The ME-PFSG assay detected A. phagocytophilum in under 35 seconds with high precision (RSD 1.30%).
- This method demonstrated increased sensitivity (200-450 pg/microL) compared to conventional slab gel electrophoresis.
- The assay required a significantly smaller sample volume (approx. 162 fL).
Conclusions:
- The developed ME-PFSG assay is a highly sensitive and rapid diagnostic tool for Anaplasma phagocytophilum in cattle.
- This method offers advantages over traditional electrophoresis, including speed and reduced sample requirements.
- The assay holds potential for effective surveillance and management of this zoonotic disease.
