Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined with a lateral flow dipstick method.

The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.

Comparative miRNA expression profile analysis of porcine ovarian follicles: new insights into the initiation mechanism of follicular atresia.

Follicular atresia occurs in every stage of ovarian development, which is relevant to female fertility. In the past decade, increasing studies have confirmed that miRNAs, a class of short non-coding RNAs, play an important role in follicular atresia by post-transcription regulation of their target genes. However, the function of miRNAs on follicular atresia initiation is unknown. In the present study, high-throughput small RNA sequencing was performed to analyze differential miRNA expression profiles between healthy (HF) follicles and early atretic (EAF) follicles. A total of 237 conserved miRNA were detected, and the miR-143 is the highest expressed in follicles. Meanwhile, we also found wide sequence variations (isomiRs) in porcine ovarian miRNA, including in 5'un-translation region, core seed sequences and 3'untranslation region. Furthermore, we identified 22 differentially expressed miRNAs in EAF groups compared to HF group, of which 3 miRNAs were upregulated, as well as 19 miRNAs were downregulated, and then the RT-PCR was performed to validate these profiles. The target genes of these differentially expressed miRNAs were predicted by using miRwalk, miRDB, and Targetscan database, respectively. Moreover, the gene ontology and KEGG pathway enrichment established that the regulating functions and signaling pathways of these miRNAs contribute to follicular atresia initiation and cell fate. In conclusion, this study provides new insights into the changes of miRNAs in early atretic follicles to demonstrate their molecular regulation in ovarian follicular atretic initiation.