Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees

• Published in: FEMS microbiology letters 2014-08, Vol.357 (1), p.40-48
• Main Authors: Ptaszyńska, Aneta A., Borsuk, Grzegorz, Woźniakowski, Grzegorz, Gnat, Sebastian, Małek, Wanda
• Format: Article
• Language: English
• Subjects: 16S rDNA gene amplification; Animals; Bees - microbiology; Biological Assay - methods; Ceramics - isolation & purification; DNA Primers - genetics; DNA, Ribosomal - genetics; LAMP; Microbiology; Microsporidia - genetics; Nosema - genetics; Nosema - isolation & purification; Nosema spp. detection; Nucleic Acid Amplification Techniques - methods; Sensitivity and Specificity; Species Specificity; Temperature
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/1574-6968.12521

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 103-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. Designed LAMP is highly Nosema species specific, 13-fold more sensitive than standard PCR and very fast - results are obtained within 3 min. Designed LAMP is highly Nosema species specific, 13-fold more sensitive than standard PCR and very fast - results are obtained within 3 min.