Optimization of duplex digital PCR for the measurement of SARS-CoV-2 RNA.

Quantitative PCR (qPCR) is the gold standard for detecting nucleic acid sequences specific to the target pathogen. For COVID-19 diagnosis, several molecular assays have been developed. In this study, we present an optimization strategy for the measurement of SARS-CoV-2 RNA via multiplex qPCR and digital PCR (dPCR). Compared to qPCR, both droplet and chip-based dPCR, which are known to be more sensitive and accurate, showed a better resilience to suboptimal assay compositions and cycling conditions following the proposed optimizations. In particular, the formation of heterodimers among assays greatly interfered with qPCR results, but only minimally with dPCR results. In dPCR, existing heterodimers lowered the PCR efficiency, producing a dampened fluorescent signal in positive partitions. This can be corrected by adjusting the PCR cycling conditions, after which dPCR shows the capability of measuring the expected copy number. In addition, we present a process to improve the existing RdRp assay by correcting the primer sequences and matching the melting temperature, ultimately producing highly sensitive and robust assays. The results of this study can reduce the cost and time of SARS-CoV-2 diagnosis while increasing accuracy. Furthermore, our results suggest that dPCR is a reliable method for the accurate measurement of nucleic acid targets.

Systematic Review and Meta-Analysis of Campylobacter Species Contamination in Poultry, Meat, and Processing Environments in South Korea.

Campylobacter spp. constitute a significant global threat as a leading cause of foodborne illnesses, with poultry meat as a prominent reservoir for these pathogens. South Korea is known for its diverse poultry consumption habits, and continuous outbreaks make it a matter of concern to perform a meta-analysis to identify the primary source of contamination. This systematic review and meta-analysis aimed to assess and compare the prevalence of Campylobacter in various poultry and meat types while also considering the importance of environmental factors in South Korea. The meta-analysis revealed that duck meat exhibited the highest prevalence of Campylobacter, with a pooled estimate of 70.46% (95% CI: 42.80% to 88.38%), followed by chicken meat at a pooled prevalence of 36.17% (95% CI: 26.44% to 47.91%). Additionally, our analysis highlighted the predominance of C. jejuni and C. coli in South Korea. These findings underscore the importance of implementing rigorous food safety measures and establishing robust surveillance programs in the poultry industry to mitigate the risk of Campylobacter-related foodborne illnesses associated with meat consumption in South Korea.

Differential Expression of Extracellular Matrix and Adhesion Molecules in Fetal-Origin Amniotic Epithelial Cells of Preeclamptic Pregnancy.

Preeclampsia is a common disease that can occur during human pregnancy and is a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia (PE). The development of this syndrome is thought to be related to multiple factors. Recently, we isolated patient-specific human amniotic epithelial cells (AECs) from the placentas of 3 women with normal pregnancy and 3 with preeclamptic pregnancy. Since the characteristics of human AECs in PE are different from those in normal pregnancy, we sought to confirm the genes differentially expressed between preeclamptic pregnancy and normal pregnancy. Therefore, we performed transcriptome analysis to investigate the candidate genes associated with the possible pathophysiology of preeclampsia. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) online resource. In this study, we selected a total of 12 pathways and focused on extracellular matrix-related and biological adhesion molecules. Using RT-PCR array and real-time PCR, we confirmed that COL16A1, ITGB2, and LAMA3 were significantly up-regulated, but ITGA1, ITGA3, ITGA6, MMP1, MMP3, MMP10 and MMP11 were significantly down-regulated in preeclamptic fetal origin cells. Taken together, we suggest that the genes and pathways identified here may be responsible for the occurrence and development of PE, and controlling their expression may play a role in communication with fetal-maternal placenta to keep normal pregnancy.