Molecular Characterization of a New Nosema bombycis Strain Detected in Iranian Silkworm Farms

Kiani-Azad, Kimia; Bagheri, Masoumeh; Sadeghi, Mostafa; Nematollahian, Shahla; Zahmatkesh, Azadeh; Moharrami, Mojtaba; Miraie-Ashtiani, Seyed Reza

doi:10.1007/s11686-022-00592-8

Cited by 2 publications

(3 citation statements)

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“…Genetic assays, such as the use of PCR and LAMP methods, have expanded the detection of pebrine disease in eggs (16). Thus, in this study, using a pair of specific primers based on a previous study (10), a detection limit of N. bombycis was determined. Findings demonstrated that the detectable amount of infection was less than 6 pg using the Real-Time PCR method, which indicated the high sensitivity of this method;…”

Section: Discussionmentioning

confidence: 99%

“…DNA was extracted from N. bombycis spores using an optimized phenol-chloroform protocol (13). Subsequently, a 136 bp fragment was selected based on the N. bombycis SSU rDNA sequence identified in a previous study (10). A pair of primers corresponding to the partial SSU rDNA fragment was designed and synthesized; F: 5ꞌ-GTCCCTGTTCTTTGTAC -3ꞌ and R: 5ꞌ-ATCCTGCTAATGGTTCT -3ꞌ.…”

Section: Dna Extraction Polymerase Chain Reactionmentioning

confidence: 99%

“…The present study aimed to diagnose pebrine disease based on molecular techniques (conventional and Real-Time PCR methods) using specific sequences resulted from previous studies (10,11). Furthermore, the estimation of the detection limit for N. bombycis infection was based on the Real-Time PCR method.…”

Section: Introductionmentioning

confidence: 99%

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Diagnosis of Pebrine Disease in Silkworm Using Molecular Methods

2022

ARI

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Dna Extraction Polymerase Chain Reactionmentioning

confidence: 99%