Diagnostic Performances of Different Genome Amplification Assays for the Detection of Swine Vesicular Disease Virus in Relation to Genomic Lineages That Circulated in Italy

During the last 25 years, swine vesicular disease (SVD) has occurred in Italy mostly sub-clinically. Therefore, regular testing of fecal samples from suspected holdings and high turnover premises was fundamental to identifying virus circulation and to achieve SVD eradication. In this study, we evaluated diagnostic performances of six genomic amplification methods, using positive fecal samples from 78 different outbreaks (1997–2014), which included different lineages. Comparison of three RT-PCRs, designed to amplify the same 154 nt portion of the gene 3D, demonstrated that a conventional and a real-time based on SYBR Green detection assay showed the highest diagnostic sensitivity, detecting all samples, while a real-time TaqMan-based test missed three cases, owing to two mismatches in the probe target sequence. Diagnostic and analytical specificities were optimal, as 300 negative field samples and other enteroviruses reacted negative. Three further evaluated tests, previously described, were a 3D-targeted reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and two real-time RT-PCRs targeted on the 5′UTR region. Here, the presence of multiple mismatches in probe and primers reduced the diagnostic performances, and two of the assays were unable to detect viruses from one sub-lineage. These results highlight that the choice of tests using less nucleotide targets significantly contributed to the success of the SVD eradication plan.

Follicular Fluid Oocyte/Cumulus-Free DNA Concentrations as a Potential Biomolecular Marker of Embryo Quality and IVF Outcome

The present prospective study examined the follicular fluid oocyte/cumulus-free DNA concentrations (ff o/c-free DNA) during ovarian stimulation and the possible association between ff o/c-free DNA and embryological results such as embryo quality and pregnancy rate. Eighty-three women undergoing IV/ICSI-ET treatments were prospectively included in this study. ff o/c-free DNA was determined by conventional quantitative real time PCR-Sybr green detection approach. The 83 ff samples were categorized in two groups: group 1n=62with cumulus oocytes complexes (CoCs)≥2 and group 2n=21with CoCs = 1. Group 1 revealed significant higher embryo quality in terms of mean score of embryo transfer (MSET), but lower ff o/c-free DNA concentrations compared to group 2. The two groups showed comparable pregnancy rates (positive hCG and clinical pregnancy). The higher the ff o/c-free DNA concentration, the lower the number of produced oocytes. ff o/c-free DNA did not seem to have any direct role in the IVF outcome. Further research is required to clarify whether ff o/c-free DNA is a biomolecular marker of embryo quality and IVF outcome.

Development and Experimental Validation of a Predictive Threshold Cycle Equation for Quantification of Virulence and Marker Genes by High-Throughput Nanoliter-Volume PCR on the OpenArray Platform

ABSTRACT Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (CT ) equation was developed to calculate the number of starting copies based on an experimental CT . The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (R 2 = 0.816) was observed between the predicted and experimental CT s based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3′ end. The performance of the predictive CT equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive CT equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.