Diagnosis of Pebrine Disease in Silkworm Using Molecular Methods.

Abstract

Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the Nosema bombycis partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of N. bombycis DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values ​​and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for N. bombycis was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.