title: Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.
abstract: Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae.
authors: Department of Botany and Mycology, Faculty of Biology and Biotechnology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland.
authors: Aneta A
authors: AA
authors: Ptaszyńska
authors: Grzegorz
authors: G
authors: Borsuk
authors: Grzegorz
authors: G
authors: Woźniakowski
authors: Sebastian
authors: S
authors: Gnat
authors: Wanda
authors: W
authors: Małek
mesh_terms: D000818:Animals; D001516:Bees / Q000382:microbiology*; D001681:Biological Assay / Q000379:methods*; D002516:Ceramics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D016814:Microsporidia / Q000235:genetics; D016818:Nosema / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013696:Temperature
chemical_list: C113675:Cerana; D017931:DNA Primers; D004275:DNA, Ribosomal
keywords: 16S rDNA gene amplification; LAMP; Nosema spp. detection