Transfusion · 2026
An in-house nucleic acid test for detection of Anaplasma phagocytophilum in whole blood donor samples.
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Kumaran Dilini, Charlton Carmen, Pambrun Chantale, Drews Steven J, Ramirez-Arcos Sandra
Аннотация
BACKGROUND: Anaplasma phagocytophilum is a tick-borne pathogen that causes anaplasmosis. Increased incidences of this disease in Canada and cases of transfusion-transmitted anaplasmosis in the United States have been reported. Currently, there are no Health Canada licensed methodologies available for detection of A. phagocytophilum nucleic acids (NAs) in blood donors. Here we describe a method for NA extraction and detection of A. phagocytophilum in whole blood (WB) donor samples.
STUDY DESIGN AND METHODS: Plasmids carrying the A. phagocytophilum msp2 and gltA genes and genomic A. phagocytophilum DNA were used to spike WB (102-1010 plasmid copies, and 3.35 × 106 genome copies/1.1 mL of WB, respectively) which was added to proprietary WB collection tubes (RWBCT). The tubes were frozen at -20°C for ≥48 h followed by NA extraction and detection of the msp2 and gltA genes using SYBR green-based qPCR. Unspiked WB and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene amplification were used as process controls. Specificity was assessed with plasmids containing gene fragments from Anaplasma marginale and Anaplasma platys. Assays were run in triplicates.
RESULTS: A. phagocytophilum msp2 and gltA were detected by qPCR in plasmid-spiked WB at a concentration of 103 copies/1.1 mL of WB; however, the gltA primer set yielded false positive results. Additionally, genomic A. phagocytophilum DNA was successfully detected in spiked WB using msp2 specific primers. Cross reaction with A. platys msp2 and A. marginale gltA was observed with the A. phagocytophilum primer sets.
CONCLUSION: A simple, scalable in-house protocol for NA extraction from RWBCT has been optimized to detect A. phagocytophilum in WB donor samples.
STUDY DESIGN AND METHODS: Plasmids carrying the A. phagocytophilum msp2 and gltA genes and genomic A. phagocytophilum DNA were used to spike WB (102-1010 plasmid copies, and 3.35 × 106 genome copies/1.1 mL of WB, respectively) which was added to proprietary WB collection tubes (RWBCT). The tubes were frozen at -20°C for ≥48 h followed by NA extraction and detection of the msp2 and gltA genes using SYBR green-based qPCR. Unspiked WB and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene amplification were used as process controls. Specificity was assessed with plasmids containing gene fragments from Anaplasma marginale and Anaplasma platys. Assays were run in triplicates.
RESULTS: A. phagocytophilum msp2 and gltA were detected by qPCR in plasmid-spiked WB at a concentration of 103 copies/1.1 mL of WB; however, the gltA primer set yielded false positive results. Additionally, genomic A. phagocytophilum DNA was successfully detected in spiked WB using msp2 specific primers. Cross reaction with A. platys msp2 and A. marginale gltA was observed with the A. phagocytophilum primer sets.
CONCLUSION: A simple, scalable in-house protocol for NA extraction from RWBCT has been optimized to detect A. phagocytophilum in WB donor samples.