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



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.

 

FEMS Microbiology Letters. 357 (1), 40–48, DOI: 10.1111/1574-6968.12521.

http://onlinelibrary.wiley.com/doi/10.1111/1574-6968.12521/abstract