AUTHOR=Sukumolanan Pratch , Demeekul Kanokwan , Petchdee Soontaree 

TITLE=Development of a Loop-Mediated Isothermal Amplification Assay Coupled With a Lateral Flow Dipstick Test for Detection of Myosin Binding Protein C3 A31P Mutation in Maine Coon Cats

JOURNAL=Frontiers in Veterinary Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.819694

DOI=10.3389/fvets.2022.819694

ISSN=2297-1769

ABSTRACT=<sec><title>Background</title><p>Myosin-binding protein C3 <italic>A31P</italic> (<italic>MYBPC3-A31P</italic>) missense mutation is a genetic deviation associated with the development of hypertrophic cardiomyopathy (HCM) in Maine Coon cats. The standard detection of the <italic>MYBPC3-A31P</italic> mutation is complicated, time-consuming, and expensive. Currently, there has been a focus on the speed and reliability of diagnostic tools. Therefore, this study aimed to develop a loop-mediated isothermal amplification assay (LAMP) coupled with a lateral flow dipstick (LFD) test to detect <italic>MYBPC3-A31P</italic> mutations in Maine Coon cats.</p></sec><sec><title>Materials and Methods</title><p>Fifty-five Maine Coon cats were enrolled in this study, and blood samples were collected. <italic>MYBPC3-A31P</italic> was genotyped by DNA sequencing. Primers for LAMP with a LFD test were designed. The optimal conditions were determined, including temperature and time to completion for the reaction. The sensitivity of <italic>A31P</italic>-LAMP detection was compared between agarose gel electrophoresis (the standard method) and the LFD test. The <italic>A31P</italic>-LAMP-LFD test was randomly performed on seven cats (four with the <italic>A31P</italic> mutation and three wild-type cats).</p></sec><sec><title>Results</title><p>The <italic>A31P</italic>-LAMP procedure was able to distinguish between cats with <italic>MYBPC3-A31P</italic> wild-type cats and <italic>MYBPC3-A31P</italic> mutant cats. The LAMP reactions were able to be completed in 60 min at a single temperature of 64◦C. Moreover, this study demonstrated that <italic>A31P</italic>-LAMP coupled with the LFD test allowed for <italic>A31P</italic> genotype detection at a lower DNA concentration than agarose gel electrophoresis.</p></sec><sec><title>Discussions</title><p>This new <italic>A31P</italic>-LAMP with a LFD test is a successful and reliable assay with a rapid method, cost-effectiveness, and low requirements for sophisticated equipment for the detection of <italic>MYBPC3-A31P</italic> mutations. Thus, this assay has excellent potential and can be recognized as a novel screening test for hypertrophic cardiomyopathy associated with <italic>MYBPC3-A31P</italic> mutations in felines.</p></sec>