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Virus screening is obligatory to avoid the spread of plant viruses regionally and globally. Double-antibody sandwich (DAS)-ELISA is the standard for screening potato viruses owing to its high-throughput potential, robustness, and cost–benefit ratio. However, low virus titers present in dormant potato tubers may not be reliably detected by using DAS-ELISA. Virus enrichment for reliable virus detection by DAS-ELISA assay is time-consuming and can be avoided by switching to more sensitive molecular biological techniques. Therefore, we developed a TaqMan® qPCR-based one-step protocol, termed direct reverse transcription quantitative PCR (DiRT-qPCR) for detection of RNA potato viruses PVY, PLRV and PVS without sophisticated nucleic acid purification and providing a high-throughput potential. Compared with DAS-ELISA, DiRT-qPCR showed up to a 100,000,000-fold higher sensitivity depending on the virus species. We also compared the qualitative results of standard DAS-ELISA used in seed potato certification, performed by sampling leaves of at least 4-weeks-old cultivated tuber eye cuttings, to the 1.5 h long DiRT-qPCR protocol on dormant tubers. The DiRT-qPCR protocol achieved an agreement with the DAS-ELISA procedure of 92.8%, 84.1% and 82.3% for the detection of PLRV, PVY, and PVS, respectively. The investigated different virus species show different multiplication behavior in secondary infected potato tuber eye cuttings, which is assumed to be a reason for the remaining qualitative differences in the outcome of the DiRT-qPCR and DAS-ELISA comparison. In our opinion, DiRT-qPCR protocol can be used as a reliable, work- and resource-saving alternative to DAS-ELISA in qualitative directed virus detection, particularly because no RNA purification is needed and dormant potato tubers can be directly used.

Early detection is invaluable for the cost‐effective control and eradication of invasive species, yet many traditional sampling techniques are ineffective at the low population abundances found at the onset of the invasion process. Environmental DNA (eDNA) is a promising and sensitive tool for early detection of some invasive species, but its efficacy has not yet been evaluated for many taxonomic groups and habitat types. We evaluated the ability of eDNA to detect the invasive rusty crayfish Orconectes rusticus and to reflect patterns of its relative abundance, in upper Midwest, USA, inland lakes. We paired conventional baited trapping as a measure of crayfish relative abundance with water samples for eDNA, which were analysed in the laboratory with a qPCR assay. We modelled detection probability for O. rusticus eDNA using relative abundance and site characteristics as covariates and also tested the relationship between eDNA copy number and O. rusticus relative abundance. We detected O. rusticus eDNA in all lakes where this species was collected by trapping, down to low relative abundances, as well as in two lakes where trap catch was zero. Detection probability of O. rusticus eDNA was well predicted by relative abundance of this species and lake water clarity. However, there was poor correspondence between eDNA copy number and O. rusticus relative abundance estimated by trap catches. Synthesis and applications. Our study demonstrates a field and laboratory protocol for eDNA monitoring of crayfish invasions, with results of statistical models that provide guidance of sampling effort and detection probabilities for researchers in other regions and systems. We propose eDNA be included as a tool in surveillance for invasive or imperilled crayfishes and other benthic arthropods.

We discuss the importance of the in vivo models in elucidating cancer biology, focusing on the patient-derived xenograft (PDX) models, which are classic and standard functional in vivo platforms for preclinical evaluation. We provide an overview of the most representative models, including cell-derived xenografts (CDX), tumor and metastatic cell-derived xenografts, and PDX models utilizing humanized mice (HM). The orthotopic models, which could reproduce the cancer environment and its progression, similar to human tumors, are particularly common. The standard procedures and rationales of gastric adenocarcinoma (GAC) orthotopic models are addressed. Despite the significant advantages of the PDX models, such as recapitulating key features of human tumors and enabling drug testing in the in vivo context, some challenges must be acknowledged, including loss of heterogeneity, selection bias, clonal evolution, stroma replacement, tumor micro-environment (TME) changes, host cell carryover and contaminations, human-to-host cell oncogenic transformation, human and host viral infections, as well as limitations for immunologic research. To compensate for these limitations, other mouse models, such as syngeneic and humanized mouse models, are currently utilized. Overall, the PDX models represent a powerful tool in cancer research, providing critical insights into tumor biology and potential therapeutic targets, but their limitations and challenges must be carefully considered for their effective use. Lastly, we present an intronic quantitative PCR (qPCR) method to authenticate, detect, and quantify human/murine cells in cell lines and PDX samples.

Conservation agriculture practices that promote soil health have distinct and lasting effects on microbial populations involved with soil nitrogen (N) cycling. In particular, using a leguminous winter cover crop (hairy vetch) promoted the expression of key functional genes involved in soil N cycling, equaling or exceeding the effects of inorganic N fertilizer. Soil microbial transformations of nitrogen (N) can be affected by soil health management practices. Here, we report in situ seasonal dynamics of the population size (gene copy abundances) and functional activity (transcript copy abundances) of five bacterial genes involved in soil N cycling (ammonia-oxidizing bacteria [AOB] amoA , nifH , nirK , nirS , and nosZ ) in a long-term continuous cotton production system under different management practices (cover crops, tillage, and inorganic N fertilization). Hairy vetch ( Vicia villosa Roth), a leguminous cover crop, most effectively promoted the expression of N cycle genes, which persisted after cover crop termination throughout the growing season. Moreover, we observed similarly high or even higher N cycle gene transcript abundances under vetch with no fertilizer as no cover crop with N fertilization throughout the cover crop peak and cotton growing seasons (April, May, and October). Further, both the gene and transcript abundances of amoA and nosZ were positively correlated to soil nitrous oxide (N 2 O) emissions. We also found that the abundances of amoA genes and transcripts both positively correlated to field and incubated net nitrification rates. Together, our results revealed relationships between microbial functional capacity and activity and in situ soil N transformations under different agricultural seasons and soil management practices. IMPORTANCE Conservation agriculture practices that promote soil health have distinct and lasting effects on microbial populations involved with soil nitrogen (N) cycling. In particular, using a leguminous winter cover crop (hairy vetch) promoted the expression of key functional genes involved in soil N cycling, equaling or exceeding the effects of inorganic N fertilizer. Hairy vetch also left a legacy on soil nutrient capacity by promoting the continued activity of N cycling microbes after cover crop termination and into the main growing season. By examining both genes and transcripts involved in soil N cycling, we showed different responses of functional capacity (i.e., gene abundances) and functional activity (i.e., transcript abundances) to agricultural seasons and management practices, adding to our understanding of the effects of soil health management practices on microbial ecology.

Oral bacteria possess the ability to form biofilms on solid surfaces. After the penetration of oral bacteria into the pulp, the contact between biofilms and pulp tissue may result in pulpitis, pulp necrosis and/or periapical lesion. Depending on the environmental conditions and the availability of nutrients in the pulp chamber and root canals, mainly Gram-negative anaerobic microorganisms predominate and form the intracanal endodontic biofilm. The objective of the present study was to investigate the role of different substrates on biofilm formation as well as the separate and collective incorporation of six endodontic pathogens, namely Enterococcus faecalis, Staphylococcus aureus, Prevotella nigrescens, Selenomonas sputigena, Parvimonas micra and Treponema denticola into a nine-species “basic biofilm”. This biofilm was formed in vitro as a standard subgingival biofilm, comprising Actinomyces oris, Veillonella dispar, Fusobacterium nucleatum, Streptococcus anginosus, Streptococcus oralis, Prevotella intermedia, Campylobacter rectus, Porphyromonas gingivalis, and Tannerella forsythia. The resulting endodontic-like biofilms were grown 64 h under the same conditions on hydroxyapatite and dentin discs. After harvesting the endodontic-like biofilms, the bacterial growth was determined using quantitative real-time PCR, were labeled using fluorescence in situ hybridization (FISH) and analyzed by confocal laser scanning microscopy (CLSM). The addition of six endodontic pathogens to the “basic biofilm” induced a decrease in the cell number of the “basic” species. Interestingly, C. rectus counts increased in biofilms containing E. faecalis, S. aureus, P. nigrescens and S. sputigena, respectively, both on hydroxyapatite and on dentin discs, whereas P. intermedia counts increased only on dentin discs by addition of E. faecalis. The growth of E. faecalis on hydroxyapatite discs and of E. faecalis and S. aureus on dentin discs were significantly higher in the biofilm containing all species than in the “basic biofilm”. Contrarily, the counts of P. nigrescens, S. sputigena and P. micra on hydroxyapatite discs as well as counts of P. micra and T. denticola on dentin discs decreased in the all-species biofilm. Overall, all bacterial species associated with endodontic infections were successfully incorporated into the standard multispecies biofilm model both on hydroxyapatite and dentin discs. Thus, future investigations on endodontic infections can rely on this newly established endodontic-like multispecies biofilm model.

Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health problems, such as waterborne outbreaks, which have caused huge economic and social losses. In this context, the prompt detection of microbial contamination becomes essential to enable early warning and timely reaction with proper interventions. Recently, molecular diagnostics have been increasingly employed for the rapid and robust assessment of microbial water quality implicated by various microbial pollutants, e.g., waterborne pathogens and antibiotic-resistance genes (ARGs), imposing the most critical health threats to humans and the environment. Continuous technological advances have led to constant improvements and expansions of molecular methods, such as conventional end-point PCR, DNA microarray, real-time quantitative PCR (qPCR), multiplex qPCR (mqPCR), loop-mediated isothermal amplification (LAMP), digital droplet PCR (ddPCR), and high-throughput next-generation DNA sequencing (HT-NGS). These state-of-the-art molecular approaches largely facilitate the surveillance of microbial water quality in diverse aquatic systems and wastewater. This review provides an up-to-date overview of the advancement of the key molecular tools frequently employed for microbial water quality assessment, with future perspectives on their applications.

Arbuscular mycorrhizal fungi (AMF) form symbioses with most terrestrial plants and are known to have a positive effect on plant growth and health. Different methodologies have been developed to assess the AMF-plant symbiosis. The most applied method, which involves staining of roots and microscopic observation of the AMF structures, is tedious and time-consuming and the results are highly dependent on the observer. Using quantitative polymerase chain reaction (qPCR) to quantify AMF root colonization represents a reliable, high-throughput technique that allows the assessment of numerous samples. Quantification with qPCR can be performed through two methods: relative quantification and absolute quantification. In relative quantification, the target gene is normalized with a reference gene. On the other hand, absolute quantification involves the use of a standard curve, for which template DNA is serially diluted. In a previous paper, we validated the primer pair AMG1F and AM1 for a relative quantification approach to assess AMF root colonization in Petunia. Here, we tested the same primers with an absolute quantification approach and compared the results with the traditional microscopy method. We evaluated the qPCR method with three different crops, namely, wheat (cv. Colmetta and Wiwa), tomato, and leek. We observed a strong correlation between microscopy and qPCR for Colmetta (r = 0.90, p < 0.001), Wiwa (r = 0.94, p < 0.001), and tomato (r = 0.93, p < 0.001), but no correlation for leek (r = 0.27, p = 0.268). This highlights the importance of testing the primer pair for each specific crop.

Quantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation. The reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen ) and QuantStudio Absolute Q (ThermoFisher ) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx ) and NGStrack™ (GenDX )) was performed. These new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity). Finally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

Arbuscular mycorrhiza fungi (AMF) are beneficial soil fungi that can promote the growth of their host plants. Accurate quantification of AMF in plant roots is important because the level of colonization is often indicative of the activity of these fungi. Root colonization is traditionally measured with microscopy methods which visualize fungal structures inside roots. Microscopy methods are labor-intensive, and results depend on the observer. In this study, we present a relative qPCR method to quantify AMF in which we normalized the AMF qPCR signal relative to a plant gene. First, we validated the primer pair AMG1F and AM1 in silico, and we show that these primers cover most AMF species present in plant roots without amplifying host DNA. Next, we compared the relative qPCR method with traditional microscopy based on a greenhouse experiment with Petunia plants that ranged from very high to very low levels of AMF root colonization. Finally, by sequencing the qPCR amplicons with MiSeq, we experimentally confirmed that the primer pair excludes plant DNA while amplifying mostly AMF. Most importantly, our relative qPCR approach was capable of discriminating quantitative differences in AMF root colonization and it strongly correlated (Spearman Rho = 0.875) with quantifications by traditional microscopy. Finally, we provide a balanced discussion about the strengths and weaknesses of microscopy and qPCR methods. In conclusion, the tested approach of relative qPCR presents a reliable alternative method to quantify AMF root colonization that is less operator-dependent than traditional microscopy and offers scalability to high-throughput analyses.

BPV1, BPV2, BPV13, and BPV14 are all genotypes of bovine delta papillomaviruses (δPV), of which the first three cause infections in horses and are associated with equine sarcoids. However, BPV14 infection has never been reported in equine species. In this study, we examined 58 fresh and thawed commercial semen samples from healthy stallions. In 34 (58.6%), bovine δPV DNA was detected and quantified using droplet digital polymerase chain reaction (ddPCR). Real time quantitative PCR (qPCR) was able to identify bovine δPV DNA in 5 samples (8.6%). Of the BPV-infected semen samples, 15 were positive for BPV2 (~ 44.1%) on ddPCR and 4 (~ 11.7%) on qPCR; 12 (~ 35.3%) for BPV14 on ddPCR and 1 (~ 3%) by qPCR; 4 (~ 11.7%) for BPV1 on ddPCR, whereas qPCR failed to reveal this infection; 3 (~ 8.8%) for BPV13 on ddPCR; and BPV13 infection was not detected by qPCR. Our study showed for the first time that BPV14 is an additional infectious agent potentially responsible for infection in horses, as its transcripts were detected and quantified in some semen samples. Large-scale BPV14 screening is necessary to provide substantial data on the molecular epidemiology for a better understanding of the geographical divergence of BPV14 prevalence in different areas and how widespread BPV14 is among equids.