Little is known about the underlying mechanisms that contribute to the persistence and degradation of DNA within soil. The goals of this study are to determine the duration of mitochondrial DNA (mtDNA) and nuclear DNA (nuDNA) persistence in soils enriched by surface-level human decomposition and to better understand the contribution of environmental factors. The surface-level decomposition of three human cadavers was documented over 11 weeks. Based on quantitative PCR results, we found nuDNA to persist in soils six weeks post-placement, while mtDNA was recoverable for the entire 11-week decomposition period. Principle components analyses and Spearman\'s rank correlations revealed that (1) time, (2) total body score, and (3) weekly average air temperature were significantly correlated with concentrations of nuDNA and mtDNA in soil, suggesting these factors play a role in the degradation of DNA in soils.

BACKGROUND: Microbiota succession determines the flavor and quality of fermented foods. Quantitative PCR-based quantitative microbiome profiling (QMP) has been applied broadly for microbial analysis from absolute abundance perspectives, transforming microbiota ratios into counts by normalizing 16S ribosomal RNA (16S rRNA) gene sequencing data with gene copies quantified by quantitative PCR. However, the application of QMP in fermented foods is still limited.
RESULTS: QMP elucidated microbial succession of Taiwanese pickled cabbage. In the spontaneous first-round fermentation (FR), the 16S rRNA gene copies of total bacteria increased from 6.1 to 10 log copies mL-1. The dominant lactic acid bacteria genera were successively Lactococcus, Leuconostoc and Lactiplantibacillus. Despite the decrease in the proportion of Lactococcus during the succession, the absolute abundance of Lactococcus still increased. In the backslopping second-round fermentation (SR), the total bacteria 16S rRNA gene copies increased from 7.6 to 9.9 log copies mL-1. The addition of backslopping starter and vinegar rapidly led to a homogenous microbial community dominated by Lactiplantibacillus. The proportion of Lactiplantibacillus remained consistently around 90% during SR, whereas its absolute abundance exhibited a continuous increase. In SR without vinegar, Leuconostoc consistently dominated the fermentation.
CONCLUSIONS: The present study highlights that compositional analysis would misinterpret microbial dynamics, whereas QMP reflected the real succession profiles and unveiled the essential role of vinegar in promoting Lactiplantibacillus dominance in backslopping fermentation of Taiwanese pickled cabbage. Quantitative microbiome profiling (QMP) was found to be a more promising approach for the detailed observation of microbiome succession in food fermentation compared to compositional analysis. © 2024 Society of Chemical Industry.

BACKGROUND: Malaria remains a severe parasitic disease, posing a significant threat to public health and hindering economic development in sub-Saharan Africa. Ethiopia, a malaria endemic country, is facing a resurgence of the disease with a steadily rising incidence. Conventional diagnostic methods, such as microscopy, have become less effective due to low parasite density, particularly among Duffy-negative human populations in Africa. To develop comprehensive control strategies, it is crucial to generate data on the distribution and clinical occurrence of Plasmodium vivax and Plasmodium falciparum infections in regions where the disease is prevalent. This study assessed Plasmodium infections and Duffy antigen genotypes in febrile patients in Ethiopia.
METHODS: Three hundred febrile patients visiting four health facilities in Jimma town of southwestern Ethiopia were randomly selected during the malaria transmission season (Apr-Oct). Sociodemographic information was collected, and microscopic examination was performed for all study participants. Plasmodium species and parasitaemia as well as the Duffy genotype were assessed by quantitative polymerase chain reaction (qPCR) for all samples. Data were analysed using Fisher\'s exact test and kappa statistics.
RESULTS: The Plasmodium infection rate by qPCR was 16% (48/300) among febrile patients, of which 19 (39.6%) were P. vivax, 25 (52.1%) were P. falciparum, and 4 (8.3%) were mixed (P. vivax and P. falciparum) infections. Among the 48 qPCR-positive samples, 39 (13%) were negative by microscopy. The results of bivariate logistic regression analysis showed that agriculture-related occupation, relapse and recurrence were significantly associated with Plasmodium infection (P < 0.001). Of the 300 febrile patients, 85 (28.3%) were Duffy negative, of whom two had P. vivax, six had P. falciparum, and one had mixed infections. Except for one patient with P. falciparum infection, Plasmodium infections in Duffy-negative individuals were all submicroscopic with low parasitaemia.
CONCLUSIONS: The present study revealed a high prevalence of submicroscopic malaria infections. Plasmodium vivax infections in Duffy-negative individuals were not detected due to low parasitaemia. In this study, an improved molecular diagnostic tool was used to detect and characterize Plasmodium infections, with the goal of quantifying P. vivax infection in Duffy-negative individuals. Advanced molecular diagnostic techniques, such as multiplex real-time PCR, loop-mediated isothermal amplification (LAMP), and CRISPR-based diagnostic methods. These techniques offer increased sensitivity, specificity, and the ability to detect low-parasite-density infections compared to the employed methodologies.

Introduction. Colorectal cancer (CRC) is a leading cause of cancer deaths, closely linked to the intestinal microbiota and bile acid metabolism. Secondary bile acids, like deoxycholic and lithocholic acid, are associated with increased CRC risk due to their disruption of vital cellular functions. In contrast, isoallolithocholic acid (isoalloLCA) shows potential health benefits, highlighting the complex role of bile acids in CRC. A specific primer set was previously developed to amplify homologs of the 5α-reductase gene (5ar), which are involved in the biosynthesis of isoalloLCA, thereby enabling the estimation of abundance of 5ar (5ar levels) in the intestine.Hypothesis/Gap Statement. We hypothesized that 5ar levels in the intestine are associated with CRC.Aim. This study aimed to investigate intestinal 5ar levels and compare them across different stages of the adenoma-carcinoma sequence, providing insights into novel strategies for monitoring CRC risk.Methodology. DNA was extracted from intestinal lavage fluids (ILF) collected during 144 colonoscopies. Next-generation sequencing (NGS) was employed to examine the sequence of 5ar homologues, using a specific primer set on DNA from seven selected ILFs - four from carcinoma patients and three from individuals with non-neoplastic mucosa. Additionally, we used quantitative PCR (qPCR) to measure 5ar levels in all 144 DNA samples.Results. We conducted 144 colonoscopies and categorized patients according to the adenoma-cancer sequence: 52 with non-neoplastic mucosa, 69 with adenomas and 23 with carcinoma. Analysis of 292,042 NGS-derived 5ar sequences revealed the seven most prevalent amplicon sequence variants, each 254 base pairs in length. These closely matched or were identical to 5ar sequences in Bacteroides uniformis, Phocaeicola vulgatus and Phocaeicola dorei. Furthermore, qPCR analysis demonstrated significantly lower 5ar levels in the carcinoma group compared to those in the non-neoplastic mucosa group (P = 0.0004). A similar, though not statistically significant, trend was observed in the adenoma group (P = 0.0763), suggesting that 5ar levels decrease as CRC progresses.Conclusion. These findings indicate that PCR-based monitoring of 5ar levels in intestinal samples over time could provide a non-invasive, rapid and cost-effective method for assessing an increased risk of CRC.

Aim: The present study aimed to figure out the potential role of exosomal microRNAs, and their targeted genes in HNC detection/diagnosis. Methods: In the present study, exosomes were extracted from the serum samples of 400 HNC patients and 400 healthy controls. Exosomes were characterized using TEM, NTA, TEM-immunogold labeling and ELISA. Quantitative PCR was used to measure the expression level of exosomal miRNA-19a, miRNA-19b and targeted genes SMAD2 and SMAD4 in HNC patients and controls. Results: The deregulation of miR-19a (p < 0.01), miR-19b (p < 0.03), SMAD2 (p < 0.04) and SMAD4 (p < 0.04) was observed in HNC patients vs controls. Conclusion: ROC curve and Kaplan-Meier analysis showed the good diagnostic/prognostic value of selected exosomal microRNAs and related genes in HNC patients.
[Box: see text].

BACKGROUND: Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments.
OBJECTIVE: A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation.
METHODS: In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method.
RESULTS: Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples.
CONCLUSIONS: The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.

Bovine digital dermatitis remains a widespread endemic disease of dairy cattle worldwide. Footbathing is commonly used as a control measure and has significant economic and environmental impact. There are few studies documenting footbathing practices on dairy farms, or evaluating their suitability for achieving foot disinfection. This study describes footbathing practices on 32 farms observed in the United Kingdom, Ireland, and the Netherlands. We measured solution depth throughout footbathing and observed levels below 7cm on 9/32 farms, which leads to inadequate foot coverage. Solution depth was associated with the number of cow passages, decreasing by 1.2cm for every 100 cow passages. We also describe levels of organic matter content (g/L) throughout footbathing as a proxy for footbath hygiene. Our data indicates that almost half of footbaths (15/32) became contaminated above the 20g/L threshold to which veterinary biocides are tested for efficacy, and that organic matter content is associated with the number of cow passages per liter of footbathing solution provided. A multivariable mixed model predicted that one liter of footbathing solution per cow should be sufficient to prevent excess contamination. As a further measure of hygiene, we tested a subset of footbath samples to quantify the amount of DNA present from the Treponema species which are considered instrumental in the etiology of digital dermatitis. We did not detect Treponema DNA in footbath samples, suggesting they are unlikely to act as infection reservoirs for this disease. Multivariable mixed models including farm identity as a random effect demonstrated that for both change in solution depth and organic matter content the effect of farm-level factors was large. Because of the magnitude of this farm effect, applying model predictions will not translate to adequate solution depth and hygiene on all farms. Our data highlights the importance of footbath auditing on individual farms.

Cytomegalovirus (CMV) colitis is a critical condition associated with severe complications in ulcerative colitis (UC). This study aimed to investigate the diagnostic value of the presence of CMV DNA in intestinal mucosa tissue and blood samples in patients with active UC. This study included 81 patients with exacerbated symptoms of UC. Patient data were obtained from the Hospital Information Management System. CMV DNA in colorectal tissue and plasma samples were analyzed using a real-time quantitative PCR assay. CMV markers were detected using immunohistochemistry and hematoxylin-eosin staining. Immunohistochemistry positivity was observed in tissue samples from eight (9.9%) patients. Only one (1.2%) patient showed CMV-specific intranuclear inclusion bodies. CMV DNA was detected in 63.0% of the tissues (median: 113 copies/mg) and in 58.5% of the plasma samples (median: 102 copies/mL). For tissues, sensitivity and the negative predictive value (NPV) for qPCR were excellent (100.0%), whereas specificity and the positive predictive value (PPV) were low (41.9% and 15.7%, respectively). For plasma, sensitivity and NPV were high (100.0%) for qPCR, whereas specificity and PPV were low (48.6% and 24.0%, respectively). CMV DNA ≥392 copies/mg in tissue samples (sensitivity 100.0% and specificity 83.6%) and ≥578 copies/mL (895 IU/mL) in plasma samples (sensitivity 66.7% and specificity 100.0%) provided an optimal diagnosis for this test. The qPCR method improved patient management through the early detection of CMV colitis in patients with UC. However, reliance on qPCR positivity alone can lead to overdiagnosis. Quantification of CMV DNA can improve diagnostic specificity, although standardization is warranted.

Seven public water systems in Minnesota, USA were analyzed from one to five times over a two-year period to assess temporal changes in the concentrations of total bacteria, Legionella spp., and Legionella pneumophila from source (i.e., raw water) through the water treatment process to the end water user. Bacterial biomass was collected by filtering large volumes of raw water (12 to 425 L, median: 38 L) or finished and tap water (27 to 1205 L, median: 448 L) using ultrafiltration membrane modules. Quantitative PCR (qPCR) was then used to enumerate all bacteria (16S rRNA gene fragments), all Legionella spp. (ssrA), and Legionella pneumophila (mip). Total coliforms, Escherichia coli, and L. pneumophila also were quantified in the water samples via cultivation. Median concentrations of total bacteria and Legionella spp. (ssrA) in raw water (8.5 and 4.3 log copies/L, respectively) decreased by about 2 log units during water treatment. The concentration of Legionella spp. (ssrA) in water collected from distribution systems inversely correlated with the total chlorine concentration for chloraminated systems significantly (p = 0.03). Although only 8 samples were collected from drinking water distribution systems using free chlorine as a residual disinfectant, these samples had significantly lower concentrations of Legionella spp. (ssrA) than samples collected from the chloraminated systems (p = 5 × 10-4). There was considerable incongruity between the results obtained via cultivation-independent (qPCR) and cultivation-dependent assays. Numerous samples were positive for L. pneumophila via cultivation, none of which tested positive for L. pneumophilia (mip) via qPCR. Conversely, a single sample tested positive for L. pneumophilia (mip) via qPCR, but this sample tested negative for L. pneumophilia via cultivation. Overall, the results suggest that conventional treatment is effective at reducing, but not eliminating, Legionella spp. from surface water supplies and that residual disinfection is effective at suppressing these organisms within drinking water distribution systems.

Diversified cropping systems and fertilization strategies were proposed to enhance the abundance and diversity of the soil microbiome, thereby stabilizing their beneficial services for maintaining soil fertility and supporting plant growth. Here, we assessed across three different long-term field experiments in Europe (Netherlands, Belgium, Northern Germany) whether diversified cropping systems and fertilization strategies also affect their functional gene abundance. Soil DNA was analyzed by quantitative PCR for quantifying bacteria, archaea and fungi as well as functional genes related to nitrogen (N) transformations; including bacterial and archaeal nitrification (amoA-bac,arch), three steps of the denitrification process (nirK, nirS and nosZ-cladeI,II) and N2 assimilation (nifH), respectively. Crop diversification and fertilization strategies generally enhanced soil total carbon (C), N and microbial abundance, but with variation between sites. Overall effects of diversified cropping systems and fertilization strategies on functional genes were much stronger than on the abundance of bacteria, archaea and fungi. The legume-based cropping systems showed great potential not only in stimulating the growth of N-fixing microorganisms but also in boosting downstream functional potentials for N cycling. The sorghum-based intercropping system suppressed soil ammonia oxidizing prokaryotes. N fertilization reduced the abundance of nitrifiers and denitrifiers except for ammonia-oxidizing bacteria, while the application of the synthetic nitrification inhibitor DMPP combined with mineral N reduced growth of both ammonia-oxidizing bacteria and archaea. In conclusion, this study demonstrates a strong impact of diversified agricultural practices on the soil microbiome and their functional potentials mediating N transformations.