Currently utilized molecular detection methods are based mainly on nucleic acid extraction, amplification, and detection procedures that may require costly equipment, numerous reagents, and highly trained personnel. These requirements make diagnostic tests expensive, time-consuming, and not suitable for point-of-care applications. There is an increasing demand for simple, low-cost portable technologies. To overcome these challenges, a paper-based elution independent collection device (EICD) was designed to collect microorganisms and recover nucleic acids for molecular biology applications with minimal steps. In this study, we demonstrate a simpler Anaplasma marginale detection that uses an EICD for nucleic acid collection combined with recombinase polymerase amplification (RPA), and a lateral flow dipstick for detection of the specified target. A pre-lysis blood treatment was optimized that uses Triton X-100 lysis buffer and bovine serum album in wash buffer. Blood samples were incubated for 5 min at room temperature and run through the EICD. Four 1-mm diameter discs excised from EICD were used as template in basic RPA and lateral flow (nfo) (endonuclease IV) RPA assays. Each disc of soluble central membrane (SCM) carried circa 0.249 pg/µl of Anaplasma DNA. The percentage of nucleic acid recoverable from the SCM ranged between 60% - 70%. Blood samples infected with A. marginale were treated with Triton X-100 pre-lysis protocol. All samples tested positive by PCR and RPA methods. EICD-driven collection of blood samples is a practical method successfully adapted to detect Anaplasma spp. or blood-borne pathogen DNA and has potential for point-of-care detection in resource-limited settings.
Keywords: Anaplasma; Blood; DNA extraction; EICD; RPA.
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