Development of a fluorescent loop-mediated isothermal amplification assay for rapid and simple diagnosis of bovine leukemia virus infection.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.

New Micro-amount of Virion Enrichment Technique (MiVET) to detect influenza A virus in the duck faeces.

Transboundary animal diseases, including highly pathogenic avian influenza, cause vast economic losses throughout the world. While it is important to identify the sources and propagation routes of the spread, such strategies are often hindered by incomplete epidemiological evidence. Isolation/detection of micro-amounts of pathogens from environmental samples is rarely successful due to the very low contamination level. This paper describes the development of the micro-amount of virion enrichment technique (MiVET), a simple and highly sensitive method that combines the use of a complex comprising a polyclonal antibody and protein G-coated magnetic beads for virion capture, and simple sodium dodecyl benzenesulfonate (SDBS) elution for low volume samples. The performance of the MiVET was evaluated using avian influenza A viruses (AIVs) in artificially spiked samples by real-time reverse transcription polymerase chain reaction (rRT-PCR). Four AIVs, H3N2, H4N2, H5N2 and H7N7, were used to artificially spike 50 ml of phosphate-buffered saline (PBS) and 1 ml of 10%-25% duck faecal supernatants. The MiVET system successfully concentrated AIVs in both PBS and faecal samples with at least 2 and 1 log greater efficacy, respectively, than conventional RNA extraction methods. The MiVET could be completed in <30 min from the beginning of sample preparation to final RNA extraction. The MiVET effectively prevented the effects of inhibitors in faecal samples, and did not require special equipment. This is the first report of this novel type of system, which is expected to be useful for the detection of micro-amounts of various veterinary and human viruses to elucidate their circulation dynamics in the environment, and for rapid and sensitive diagnosis with greater detection power.