Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

In the current pandemic, the presence of SARS-CoV-2 RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ infectiveness. Recently, the demand for laboratory reagents has greatly increased; in particular, there is a worldwide shortage of RNA extraction kits. Here, we describe a fast, simple and inexpensive method for the detection of SARS CoV-2 RNA, which includes a pretreatment with Proteinase K and a heating-cooling cycle before the amplification. This method bypasses the RNA extraction step; it leads to a higher amount of available viral RNA compared to the automated extraction methods, and generates the same profile in the subsequent amplification phase.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3–7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2’-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

KRAS somatic mutations are found in 30–40% of colorectal cancer (CRC). Seven mutations in codons 12 and 13 of KRAS (95% of the observed human mutations) preclude the efficacy of anti-EGFR therapy for the treatment of CRC. Assessment of KRAS mutational status has become a standard procedure in the management of patients with CRC. Technically, KRAS mutation testing can be performed with different methods, characterized by distinct sensitivities and specificities. The present study analyzed KRAS in 182 CRC histological samples by using direct sequencing and a new kit based on a Real-Time Sequence-Specific Primers-PCR technology. The kit allowed to recover as positive 17 samples that were negative or unclear by sequencing, with a recovery rate equal to 13.82%. This study proposes a fast, sensitive, and high-throughput system to identify such seven described mutations of KRAS gene in CRC samples.

Background: An assessment of viral load in biologic specimens of subjects with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may have important implications for public health planning. The aim of this study was to estimate the prevalence of high viral load in upper respiratory specimens of patients with SARS-CoV-2 infection during the first Italian wave (spring) and at the beginning of the second wave (summer) of the COVID-19 epidemic, through the measurement of cycle threshold (Ct) values from real-time reverse transcription-polymerase chain reaction tests conducted at the University Hospital of Udine, Italy, serving 530,000 inhabitants. Methods: We compared the prevalence of high viral load, defined as Ct ≤ 20 at the first positive test result, in 262 subjects from the spring and 453 from the summer period. Logistic regression was used to account for potential confounding due to sex, age, and severity of infection. Results: In the spring, 9.2% of subjects had Ct ≤ 20 versus 21.4% in the summer. After adjusting for confounders, the likelihood of having high viral load was 2.9 times higher in the summer than in the spring (95% confidence interval, 1.7–5.0). Conclusions: In this Italian area, more COVID-19 patients had high viral load in the spring epidemic wave than at the beginning of the second, during the summer. Cycle threshold values may represent useful information to monitor viral load at a population level in subjects with SARS-CoV-2 infection.

Severe maternal and newborn morbidity and mortality associated with pre-eclampsia, which are caused partly by premature delivery, affect a factual proportion of pregnancies. Despite its prevalence, the underlying causes of pre-eclampsia remain elusive, with emerging evidence implicating the aryl hydrocarbon receptor (AhR) in its pathogenesis. This study sought to elucidate the involvement of the AhR and its associated pathway in pre-eclampsia by comparing placental components of the AhR pathway in pregnant individuals with and without pre-eclampsia. This case–control investigation was conducted at the University Hospital of Udine from May 2021 to February 2023. The AhR was assessed using immunohistochemistry and immunofluorescence, and its mRNA was evaluated using a Real-Time Quantitative Reverse Transcription PCR. Levels of mRNA expression were also estimated for other components of the AhR pathway (CYP1B1, IDO1, ARNT, TIPARP, S100A4, and AHRR). Our findings show decreased levels of expression of AhR, IDO1, ARNT, TiPARP, and S100A4 in the placental tissues of individuals with pre-eclampsia compared to controls (p < 0.05). The AhR exhibited a distinct localization within the syncytiotrophoblast (nuclei and cytoplasm) and CD45-positive cells (nuclei and cytoplasm). Furthermore, a significant positive correlation between the AhR and S100A4 (rho = 0.81) was observed in normal placentas, while CYP1B1 displayed a significant negative correlation with the AhR (rho = −0.72), within addition to its negative correlation with TiPARP (rho = −0.83). This study illuminates pre-eclampsia’s molecular aberrations, suggesting new diagnostic, therapeutic, and mechanistic approaches. This study emphasizes the need for more research to validate and broaden these findings to improve the management of this complex pregnancy condition.