Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Listeria monocytogenes in Cooked Ham.

Changing eating habits and rising demand of food have increased the incidence of foodborne diseases, particularly in industrialized countries. In this context, contaminated ready-to-eat food (RTE) may be a vehicle for the transmission of Listeria monocytogenes (L. monocytogenes), a foodborne pathogen responsible of listeriosis, a severe infectious disease involving humans and animals. It would be useful to have rapid detection methods to screen the presence of L. monocytogenes in food. In this study, a colorimetric Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of L. monocytogenes in 37 experimentally contaminated RTE meat samples. The LAMP primers consisted of a set of six primers targeting eight regions on the hlyA gene; the assay was carried out in 30 min at 65 °C in a water bath. Amplification products were visualized by color change assessment. The results of colorimetric LAMP assays based on the hly gene obtained in this study were compared to microbiological cultural methods, real-time PCR and real-time LAMP PCR, which show 100% specificity and sensitivity. These data suggest that colorimetric LAMP assays can be used as a screen to detect L. monocytogenes in ready-to-eat meat food.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Bovine viral diarrhoea virus 1b infection associated with congenital tremor and hypomyelination in Holstein calves.

Hypomyelination is a rare consequence of in utero bovine viral diarrhoea virus (BVDV) infection. We describe a BVDV outbreak in a naïve Holstein dairy herd in northern Italy, with an unusually high prevalence of calves with neurological signs, generalised tremors and ataxia. Histological analysis showed that hypomyelination was the predominant lesion and that the most typical BVDV neuropathological findings (e.g. cerebellar hypoplasia) were absent. Virological and molecular analyses showed that non-cytopathic BVDV genotype 1b was associated with the calves' neurological signs and excluded other viruses responsible for congenital infection or neurological disorders. Whole-genome sequencing of BVDVs from the brain of a calf with neurological signs and the whole blood of a persistently infected herd-mate with no such sign showed >99.7 % sequence identity. Analysis of the quasispecies distribution revealed the greatest variation rates in regions coding for the structural proteins E1 and E2. Variation was slightly greater in the brain- than in the blood-derived sequence and occurred at different sites, suggesting the occurrence of distinct evolutionary processes in the two persistently infected calves. Molecular characterisation of BVDV genomes from five other calves with neurological signs from the same farm confirmed that the E1 and E2 regions were the most variable. Several factors, including genetic variability and host factors, appear to have contributed to the observed unique BVDV disease phenotype, characterised by hypomyelination and neurological signs.