Development and evaluation of loop-mediated isothermal amplification for rapid detection of Nosema ceranae in honeybee

Abstract

© 2016 Asian Pacific Tropical Medicine Press Objective To develop loop-mediated isothermal amplification (LAMP) to detect Nosema ceranae (N. ceranae) in honeybee samples. Methods LAMP primers were designed recognizing six distinct fragments of 16s rRNA gene and LAMP reaction was determined by optimizing the concentration of reagents, such as forward inner primer and backward inner primer, deoxynucleoside triphosphate and betaine, time and temperature. Ten-fold serial dilutions of DNA were used to determine the detection limit and accuracy using both LAMP and PCR tests. Results LAMP required 1.2 μmol/L of forward inner primer and backward inner primer primers, 0.2 μmol/L of forward outer primers and backward outer primer, 2 μmol/L of Mg2+, 0.6 mol/L of betaine, 0.6 μmol/L of deoxynucleoside triphosphate, 4.8 IU of Bst DNA polymerase and 30 ng of DNA. The optimal temperature was 63 °C and after a 40-min incubation time, a clearly ladder-like pattern of LAMP product appeared in the gel electrophoresis. LAMP appeared more sensitive than a standard PCR in detection of N. ceranae. Conclusions LAMP gave a good results and it could be an alternative diagnostic tool instead of PCR to detect N. ceranae infection in honeybee.