Recompress rpa6/5/2023 ![]() ![]() Given the complex nature of Anaplasma infections and the current lack of point-of-care diagnostic tools, specific, sensitive, easy-to-use, and rapid isothermal detection assays are needed to improve the accuracy of Anaplasma diagnosis in livestock worldwide. Therefore, RPA can be applied directly in point-of-care diagnostic center or resource-limited areas while PCR or ELISA based analyses require expensive laboratory equipment and trained personnel. Additionally, RPA reagents can be stored for 3 weeks at room temperature without need for low-temperature storage because they are stable as lyophilized pellets 21. The advantage of RPA is the minimal investment for equipment due to low reaction temperatures (25–45 ☌), relatively short incubation periods (20–40 min) and use of sensitive and specific primers and probe 20. Developed in 2006, RPA represents an innovative isothermal amplification technology that has been used to detect a range of pathogens in agriculture, human and veterinary medicine, as well as food safety 19, 20. marginale 14, 15, 16, 17, 18 but, to date, they are only being used in laboratory-based settings. LAMP assays have been developed to detect A. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are isothermal methods that are being used to improve cost and time, and issues encountered by DNA-based diagnostics 13. A variety of polymerase chain reaction (PCR) assays exist that detect Anaplasma species but the low limit of detection for early and chronic infections, cost of the equipment needed to run the assay, and the need for trained personnel limits its effectiveness in point-of-care conditions 2, 9, 10, 11, 12. marginale detection in cattle is a USDA-approved cELISA, a serologic test that targets a protein epitope of the highly conserved Anaplasma msp5 gene but it does not distinguish among species 6, 7. Detection methods for these pathogens include a wide variety of microscopy, antibody-based, and molecular methods 2, 6, 7, 8. The three species of concern are Anaplasma marginale, A. Anaplasmosis is caused by bacterium in the genus Anaplasma species and infects a broad range of animals such as cattle, sheep, and humans 5. Ticks are blood-sucking arthropods that transmit a wide variety of pathogens like viruses, bacteria, and protozoa 4, including Anaplasma species. causing losses of more than $300 million per year while in Latin America, the losses are estimated to be approximately $800 million per year 3. Increased mortality and morbidity due to bovine anaplasmosis affects the economy of the U.S. is bovine anaplasmosis which causes significant financial losses for producers 2, 3. One of the main tick-borne diseases impacting the cattle industry in the U.S. In 2017, cattle production was the most important livestock industry in the United States with an approximate value of $50.2 billon, followed by poultry ($42.7 billion) and swine production ($19.2 billion) 1. Livestock production in the United States is a significant part of the economy. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited. marginale-positive serum samples, and all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA- nfo assays detected all A. phagocytophilum, and 5.51 × 10 3 copies/µl of internal control (GAPDH). phagocytophilum, and for each multiplex lateral flow or RPA- nfo assays is 8.99 × 10 3 copies/µl of A. The limit of detection of gel-based or RPA-basic assays is 8.99 × 10 4 copies/µl = A. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. ![]()
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