Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Anisakis spp. DNA in Processed Fish Products.

Parasites belonging to the Anisakis genera are organisms of interest for human health because they are responsible for the Anisakiasis zoonosis, caused by the ingestion of raw or undercooked fish. Furthermore, several authors have reported this parasite to be a relevant inducer of acute or chronic allergic diseases. In this work, a rapid commercial system based on Loop-Mediated Isothermal Amplification (LAMP) was optimised and validated for the sensitive and rapid detection of Anisakis spp. DNA in processed fish products. The specificity and sensitivity of the LAMP assay for processed fish samples experimentally infected with Anisakis spp. larvae and DNA were determined. The LAMP system proposed in this study was able to give positive amplification for all the processed fish samples artificially contaminated with Anisakis spp., giving sensitivity values equal to 100%. Specificity tests provided no amplification for the Contracaecum, Pseudoterranova, or Hysterothylacium genera and uninfected samples. The limit of detection (LOD) of the LAMP assay proposed was 102 times lower than the real-time PCR method compared. To the best of our knowledge, this is the first report regarding the application of the LAMP assay for the detection of Anisakis spp. in processed fish products. The results obtained indicate that the LAMP assay validated in this work could be a reliable, easy-to-use, and convenient tool for the rapid detection of Anisakis DNA in fish product inspection.

Improvement of a rapid direct blood culture microbial identification protocol using MALDI-TOF MS and performance comparison with SepsiTyper kit.

Fast diagnosis of pathogens is critical to guarantee the most adequate therapy for infections; bacterial culture methods, which constitute the actual gold standard, are precise and sensitive but rather slow. Today, new methods have been made available to enable faster diagnosis, with the Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS) technique being the most promising. Even if simpler and faster than traditional bacterial culture methods, analysis of positive blood cultures via MALDI-TOF MS requires a preliminary extraction process of samples. In this study, we compared two extraction protocols for bacterial identification directly from positive blood cultures using the Bruker MALDI Biotyper system (Bruker Daltonics, Billerica, MA). In particular, we evaluated the time employed and the overall performance for their accurate identification. In this work, the performances of a commercial extraction kit, named SepsiTyper™ Kit, and those of the protocol developed by Treibmann et al. were evaluated and proven to be similar. However, the SELTERS method represents the best compromise price/performance. Lastly, an in-house developed analysis protocol has been tested, and the introduced optimizations granted a performance level equal if not better than the SepsiTyper kit, a reduced processing time and reduced costs.

Evaluation of a loop-mediated isothermal amplification method for the detection of Listeria monocytogenes in dairy food.

Objective of the present study was to test the performances of a loop-mediated isothermal amplification (LAMP)-based method for the detection of Listeria monocytogenes, with particular focus on the dairy products. The specificity of the method was evaluated on 42 different Listeria spp. strains from collections, food and environmental samples. 100% (32 of 32) of the L. monocytogenes strains were correctly recognised, and none of other 10 Listeria spp. strains was misidentified. The sensitivity was evaluated on four L. monocytogenes strains from different sources. The instrument was able to detect 10-400 CFU/mL. The ability to detect low initial numbers of L. monocytogenes (0.3-0.7 Log CFU/g) was also evaluated, in duplicate, in pasteurised milk (whole and skimmed) and dairy samples (fresh ricotta, crescenza, mascarpone, mozzarella, cottage cheese, cream cheese, taleggio, gorgonzola). The analysis was performed after 18, 24 and 48 h of incubation, and was coupled with the count of L. monocytogenes in the broth. Microbial loads were insufficient to achieve a positive result after 18 and 24 h in most of the samples; after 48 h, all the products, except taleggio and one gorgonzola sample, were identified as positive; the sensitivity of the method when applied to contaminated dairy foods was about 5 Log CFU/g. The LAMP method tested can be considered a very useful tool, as it is a costeffective and easy-functioning method. The preliminary data obtained should be confirmed with a validation process taking into account different food typologies.

Identification of embryo-fetal cells in celomic fluid using morphological and short-tandem repeats analysis.

OBJECTIVE: The main problem to wide acceptability of celocentesis as earlier prenatal diagnosis is contamination of the sample by maternal cells. The objective of this study was to investigate the cellular composition of celomic fluid for morphological discrimination between maternal and embryo-fetal cells. METHOD: Celomic fluids were aspired by ultrasound-guided transcervical celocentesis at 7-9 weeks' gestation from singleton pregnancies before surgical termination for psychological reasons. DNA extracted from celomic fluid cells showed the same morphology, and quantitative fluorescent polymerase chain reaction (PCR) assay was performed to evaluate their fetal or maternal origin. RESULTS: Six different types of non-hematological maternal and four different types of embryo-fetal cells were detected. The most common maternal cells were of epithelial origin. The majority of embryo-fetal cells were roundish with a nucleus located in an eccentric position near the wall. These cells were considered to be erythroblasts, probably derived from the yolk sac that serves as the initial site of erythropoiesis. CONCLUSIONS: The combined use of morphology and DNA analysis makes it possible to select and isolate embryo-fetal cells, even when maternal contamination is high. This development provides the opportunity for the use of celocentesis for early prenatal diagnosis of genetic diseases and application of array comparative genomic hybridization. © 2016 John Wiley & Sons, Ltd.