Application and Evaluation of LAMP-PCR for the Diagnosis of Silkworm Pebrine Disease
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For stable silkworm breeding and high-quality sericulture product production, the detection of Pebrine disease in silkworm eggs is critical. Current diagnostic methods can be timeconsuming and complex. This study aimed to develop a simplified and rapid diagnostic method using loop-mediated isothermal amplification (LAMP) technology to detect pebrine infection in silkworm mother moths. Eight primer candidates targeting the ribosomal gene region of microsporidia were designed and evaluated for specificity and detection sensitivity. A simplified nucleic acid extraction method was established, and isothermal amplification was performed using the selected primers. Of these, primers ID30 and ID45 showed no polymerization, while ID5, ID18, and ID76 exhibited nonspecific reactions, making them unsuitable. Primers ID1, ID6, ID45, and ID82 successfully amplified DNA only in the presence of pebrine, with ID82 demonstrating the best reproducibility and sensitivity, detecting as low as 2.5 pg/ul of DNA through electrophoresis and 5 pg/ul via a colorimetric change with phenol red. The entire process, from nucleic acid extraction to detection, was completed within 60 min. The use of the ID82 primer set in LAMP technology offers a promising and efficient approach for the rapid diagnosis of pebrine disease, potentially enhancing quality control in sericulture.
Abstract
For stable silkworm breeding and high-quality sericulture product production, the detection of Pebrine disease in silkworm eggs is critical. Current diagnostic methods can be timeconsuming and complex. This study aimed to develop a simplified and rapid diagnostic method using loop-mediated isothermal amplification (LAMP) technology to detect pebrine infection in silkworm mother moths. Eight primer candidates targeting the ribosomal gene region of microsporidia were designed and evaluated for specificity and detection sensitivity. A simplified nucleic acid extraction method was established, and isothermal amplification was performed using the selected primers. Of these, primers ID30 and ID45 showed no polymerization, while ID5, ID18, and ID76 exhibited nonspecific reactions, making them unsuitable. Primers ID1, ID6, ID45, and ID82 successfully amplified DNA only in the presence of pebrine, with ID82 demonstrating the best reproducibility and sensitivity, detecting as low as 2.5 pg/ul of DNA through electrophoresis and 5 pg/ul via a colorimetric change with phenol red. The entire process, from nucleic acid extraction to detection, was completed within 60 min. The use of the ID82 primer set in LAMP technology offers a promising and efficient approach for the rapid diagnosis of pebrine disease, potentially enhancing quality control in sericulture.
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