The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens.

Foodborne pathogens continue to be a major health concern worldwide. Culture-dependent methodologies are still considered the gold standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor intensive. The implementation of real-time PCR has allowed to overcome these limitations, and even reduce the cost associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter, a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopy Observation.

Foodborne pathogens are responsible for foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can be entered in a viable but nonculturable (VBNC) state. VBNC cells are characterized by an active metabolism and a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples and thus may pose a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method combining propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.

Comparing Gene Expression Between Planktonic and Biofilm Cells of Foodborne Bacterial Pathogens Through RT-qPCR.

Like most microorganisms, important foodborne pathogenic bacteria, such as Salmonella enterica, Listeria monocytogenes, and several others as well, can attach to surfaces, of either abiotic or biotic nature, and create biofilms on them, provided the existence of supportive environmental conditions (e.g., permissive growth temperature, adequate humidity, and nutrient presence). Inside those sessile communities, the enclosed bacteria typically present a gene expression profile that differs from the one that would be displayed by the same cells growing planktonically in liquid media (free-swimming cells). This altered gene expression has important consequences on cellular physiology and behavior, including stress tolerance and induction of virulence. In this chapter, the methodology to use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to monitor and comparatively quantify expression changes in preselected genes of bacteria between planktonic and biofilm growth modes is presented.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Quantitative real time PCR for distinction between Pneumocystis jirovecii infection/colonization in hospitalized patients.

Background: Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency. Methods: To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)-rRNA gene. Results: Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probe-based qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: ≤32 as infection, >35 as colonization, and a grey zone between these values (32 < X ≤ 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR). Conclusions: This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions.

Detection and characterization of Campylobacter in air samples from poultry houses using shot-gun metagenomics - a pilot study.

BACKGROUND: Foodborne pathogens such as Campylobacter jejuni are responsible for a large proportion of the gastrointestinal infections worldwide associated with poultry meat. Campylobacter spp. can be found in the chicken fecal microbiome and can contaminate poultry meat during the slaughter process. Commonly used sampling methods to detect Campylobacter spp. at poultry farms use fecal droppings or boot swabs in combination with conventional culture techniques or PCR. In this pilot study, we have used air filtering and filters spiked with mock communities in combination with shotgun metagenomics to detect Campylobacter and test the applicability of this approach for the detection and characterization of foodborne pathogens. To the best of our knowledge is this the first study that combines air filtering with shotgun metagenomic sequencing for detection and characterization of Campylobacter. RESULTS: Analysis of air filters spiked with different levels of Campylobacter, into a background of mock or poultry house communities, indicated that we could detect as little as 200 colony forming units (CFU) Campylobacter per sample using our protocols. The results indicate that even with limited sequencing effort we could detect Campylobacter in the samples analysed in this study. We observed significant amounts of Campylobacter in real-life samples from poultry houses using both real-time PCR as well as shotgun metagenomics, suggesting that the flocks in both houses were infected with Campylobacter spp. Interestingly, in both houses we find diverse microbial communities present in the indoor air which reflect the fecal microbiome of poultry. Some of the identified genera such as Staphylococcus, Escherichia and Pseudomonas are known to contain opportunistic pathogenic species. CONCLUSIONS: These results show that air sampling of poultry houses in combination with shotgun metagenomics can detect and identify Campylobacter spp. present at low levels. This is important since early detection of Campylobacter enables measures to be put in place to ensure the safety of broiler products, animal health and public health. This approach has the potential to detect any pathogen present in poultry house air.

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutLα that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and ß-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 ± 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

Laboratory-based molecular test alternatives to RT-PCR for the diagnosis of SARS-CoV-2 infection.

BACKGROUND: Diagnosing people with a SARS-CoV-2 infection played a critical role in managing the COVID-19 pandemic and remains a priority for the transition to long-term management of COVID-19. Initial shortages of extraction and reverse transcription polymerase chain reaction (RT-PCR) reagents impaired the desired upscaling of testing in many countries, which led to the search for alternatives to RNA extraction/purification and RT-PCR testing. Reference standard methods for diagnosing the presence of SARS-CoV-2 infection rely primarily on real-time reverse transcription-polymerase chain reaction (RT-PCR). Alternatives to RT-PCR could, if sufficiently accurate, have a positive impact by expanding the range of diagnostic tools available for the timely identification of people infected by SARS-CoV-2, access to testing and the use of resources. OBJECTIVES: To assess the diagnostic accuracy of alternative (to RT-PCR assays) laboratory-based molecular tests for diagnosing SARS-CoV-2 infection. SEARCH METHODS: We searched the COVID-19 Open Access Project living evidence database from the University of Bern until 30 September 2020 and the WHO COVID-19 Research Database until 31 October 2022. We did not apply language restrictions. SELECTION CRITERIA: We included studies of people with suspected or known SARS-CoV-2 infection, or where tests were used to screen for infection, and studies evaluating commercially developed laboratory-based molecular tests for the diagnosis of SARS-CoV-2 infection considered as alternatives to RT-PCR testing. We also included all reference standards to define the presence or absence of SARS-CoV-2, including RT-PCR tests and established clinical diagnostic criteria. DATA COLLECTION AND ANALYSIS: Two authors independently screened studies and resolved disagreements by discussing them with a third author. Two authors independently extracted data and assessed the risk of bias and applicability of the studies using the QUADAS-2 tool. We presented sensitivity and specificity, with 95% confidence intervals (CIs), for each test using paired forest plots and summarised results using average sensitivity and specificity using a bivariate random-effects meta-analysis. We illustrated the findings per index test category and assay brand compared to the WHO's acceptable sensitivity and specificity threshold for diagnosing SARS-CoV-2 infection using nucleic acid tests. MAIN RESULTS: We included data from 64 studies reporting 94 cohorts of participants and 105 index test evaluations, with 74,753 samples and 7517 confirmed SARS-CoV-2 cases. We did not identify any published or preprint reports of accuracy for a considerable number of commercially produced NAAT assays. Most cohorts were judged at unclear or high risk of bias in more than three QUADAS-2 domains. Around half of the cohorts were considered at high risk of selection bias because of recruitment based on COVID status. Three quarters of 94 cohorts were at high risk of bias in the reference standard domain because of reliance on a single RT-PCR result to determine the absence of SARS-CoV-2 infection or were at unclear risk of bias due to a lack of clarity about the time interval between the index test assessment and the reference standard, the number of missing results, or the absence of a participant flow diagram. For index tests categories with four or more evaluations and when summary estimations were possible, we found that: a) For RT-PCR assays designed to omit/adapt RNA extraction/purification, the average sensitivity was 95.1% (95% CI 91.1% to 97.3%), and the average specificity was 99.7% (95% CI 98.5% to 99.9%; based on 27 evaluations, 2834 samples and 1178 SARS-CoV-2 cases); b) For RT-LAMP assays, the average sensitivity was 88.4% (95% CI 83.1% to 92.2%), and the average specificity was 99.7% (95% CI 98.7% to 99.9%; 24 evaluations, 29,496 samples and 2255 SARS-CoV-2 cases); c) for TMA assays, the average sensitivity was 97.6% (95% CI 95.2% to 98.8%), and the average specificity was 99.4% (95% CI 94.9% to 99.9%; 14 evaluations, 2196 samples and 942 SARS-CoV-2 cases); d) for digital PCR assays, the average sensitivity was 98.5% (95% CI 95.2% to 99.5%), and the average specificity was 91.4% (95% CI 60.4% to 98.7%; five evaluations, 703 samples and 354 SARS-CoV-2 cases); e) for RT-LAMP assays omitting/adapting RNA extraction, the average sensitivity was 73.1% (95% CI 58.4% to 84%), and the average specificity was 100% (95% CI 98% to 100%; 24 evaluations, 14,342 samples and 1502 SARS-CoV-2 cases). Only two index test categories fulfil the WHO-acceptable sensitivity and specificity requirements for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. In addition, WHO-acceptable performance criteria were met for two assays out of 35 when tests were used according to manufacturer instructions. At 5% prevalence using a cohort of 1000 people suspected of SARS-CoV-2 infection, the positive predictive value of RT-PCR assays omitting/adapting RNA extraction/purification will be 94%, with three in 51 positive results being false positives, and around two missed cases. For TMA assays, the positive predictive value of RT-PCR assays will be 89%, with 6 in 55 positive results being false positives, and around one missed case. AUTHORS' CONCLUSIONS: Alternative laboratory-based molecular tests aim to enhance testing capacity in different ways, such as reducing the time, steps and resources needed to obtain valid results. Several index test technologies with these potential advantages have not been evaluated or have been assessed by only a few studies of limited methodological quality, so the performance of these kits was undetermined. Only two index test categories with enough evaluations for meta-analysis fulfil the WHO set of acceptable accuracy standards for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. These assays might prove to be suitable alternatives to RT-PCR for identifying people infected by SARS-CoV-2, especially when the alternative would be not having access to testing. However, these findings need to be interpreted and used with caution because of several limitations in the evidence, including reliance on retrospective samples without information about the symptom status of participants and the timing of assessment. No extrapolation of found accuracy data for these two alternatives to any test brands using the same techniques can be made as, for both groups, one test brand with high accuracy was overrepresented with 21/26 and 12/14 included studies, respectively. Although we used a comprehensive search and had broad eligibility criteria to include a wide range of tests that could be alternatives to RT-PCR methods, further research is needed to assess the performance of alternative COVID-19 tests and their role in pandemic management.

Absence of measurable quantities of Candida auris and Cryptococcus spp. in the gut microbiota of Ghanaian individuals with and without HIV infection as confirmed by applying multiple real-time PCR assays.

Introduction. Fungal infections are relevant health risks for individuals with acquired immunodeficiency in the resource-limited tropics, but available surveillance data are scarce. For Candida auris and Cryptococcus spp., the evolution from environmental reservoirs to human pathogens causing life-threatening diseases is currently discussed as a public health concern in the context of climate change and limited treatment options.Gap statement. Uncovering the gastrointestinal tract as an epidemiological niche of fungi emerging from the environment into individuals for whom fungal infections are not diagnosed.Aim. To contribute to data on the local epidemiology of C. auris and Cryptococcus spp. in Western African Ghana by analysing gastrointestinal samples of Ghanaian individuals.Methodology. Four real-time PCR assays targeting C. auris and five real-time PCR assays targeting Cryptococcus spp. were applied with stool samples of 875 non-age-stratified Ghanaian HIV patients and 30 Ghanaian control individuals without known HIV infection. Also, 664 samples from Ghanaian children under 2 years of age were investigated. The true abundance of the target micro-organism was considered as unlikely in the case of one or fewer positive signals, likely in the case of two to three positive signals and highly likely in the case of four or more positive signals per sample in the real-time PCR assays.Results. The combined application of sensitive, target-specific real-time PCR assays indicates that neither C. auris, Cryptococcus neoformans complex nor Cryptococcus gattii complex were part of the gut microbiota of Ghanaian individuals with or without HIV infection.Conclusion. Despite the significant disease burden from these pathogens in immunosuppressed Ghanaian individuals, detection from gastrointestinal samples was unlikely, which should be taken into account when discussing screening strategies for these fungi of public health concern. In contrast, the detection of these fungi from such samples should not routinely be considered as commensal colonization flora.

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale.

BACKGROUND: The parasite species Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), formerly known as Plasmodium ovale, are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species. METHODS: Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC. RESULTS: The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/µl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/µl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/µl for P. ovalecurtisi and P. ovalewallikeri, respectively. CONCLUSIONS: We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.

Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC).

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging. This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking. Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research. In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

RGeasy: a reference gene analysis tool for gene expression studies via RT-qPCR.

Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.

Real-time qPCR coupled with high-resolution melting curve analysis for the detection of the internal transcribed spacer 1 of Angiostrongylus costaricensis.

Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.

Development and validation of a droplet digital PCR assay for Nipah virus quantitation.

BACKGROUND: Nipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV. RESULTS: Our RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads. CONCLUSIONS: These findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

PPIA, HRPT1, and PGK1 genes as the appropriate combination for RT-qPCR normalization in alveolar and femoral bone remodeling in olanzapine-treated rats.

Reliable gene expression analysis in bone remodeling studies requires an appropriate selection of internal controls, i.e. stable reference genes for the normalization of quantitative real-time PCR (RT-qPCR), the most common method used for quantifying gene expression measurements. Even the most widely used reference genes can have variable expression under different experimental conditions, or in different tissue types or treatment regimes, so selecting appropriate controls is a key step in ensuring reliable results. The aim of this research was to identify the most stable reference gene(s) for the study of olanzapine modulated bone remodeling in rats. RNA was isolated from the maxillary alveolar and femoral bones of olanzapine or placebo-treated Wistar rats and transcribed to cDNA. The expression of 12 candidate reference genes was assessed by RT-qPCR. Their expressions were analysed using GeNorm, NormFinder, BestKeeper and delta Ct algorithms, and by the comprehensive ranking method. PPIA, HRPT1 and PGK1 were the most stably expres sed reference genes and the combination of the three genes was optimal for normalization. This study is the first to identify the optimal reference genes for research in olanzapine-exposed rats, which serve as a pivotal benchmark for enhancing the accuracy and reliability of future RT-qPCR expression in bone studies.

Evaluation of an in-house pan-Malassezia quantitative PCR in human clinical samples.

Althought Malassezia spp. have been involved in various pathologies, they are an integral part of the cutaneous, gut, oral, ears, nose and throat (ENT) mycobiota. Since Malassezia are difficult to grow in culture, unexhaustive molecular biology methods have been developed to detect them. The aim of the study was to evaluate an in-house pan-Malassezia quantitative polymerase chain reaction (panM-qPCR) on various clinical human samples and determine Malassezia burden in various human mycobiota. The panM-qPCR was designed to target the repeated 28S rDNA gene from all Malassezia species. We used the assay to quantify the Malassezia burden on 361 samples from 161 subjects (80 skin swabs from 10 healthy volunteers (HV), 13 samples from 2 seborrheic dermatitis patients (SD), 90 skin samples from 19 burned patients, 119 stool samples from 89 immunocompromised patients, 59 ENT samples from 41 patients). For HV, the amount of Malassezia was different according to the swabbed areas. Quantification cycle (Cq) in SD is lower than in HV. In burned patients, Cq was significantly lower compared to HV. In stool samples, 6.7% were positive for Malassezia spp. with a high Cq. For the ENT area, a higher proportion of positive specimens were detected in ear samples than in nose samples. Our findings emphasized the importance of qPCR, confirming elevated Malassezia spp. levels on individuals' faces and scalps, increased burden in SD patients and in severely burnt patients than in HV. The pan-MqPCR appears to be a promising tool for studying Malassezia in various human mycobiota.

Malassezia species are ubiquitous members of various human microbiomes, including cutaneous and mucosal sites. While these fungi have been implicated in several pathologies, their presence as commensals complicates their study, especially due to difficulties in culturing them in vitro. This has necessitated the development of molecular techniques to detect and quantify Malassezia species directly from clinical samples. In this study, we report on the development and application of an in-house pan-Malassezia quantitative PCR (panM-qPCR) assay. This assay targets the conserved 28S rDNA gene across all known Malassezia species, allowing for a broad-spectrum detection approach. We applied this panM-qPCR to a diverse set of clinical samples, totaling 361 specimens from 161 subjects, encompassing healthy individuals, patients with seborrheic dermatitis, burn victims, and immunocompromised individuals. Our results indicate variable Malassezia loads on different skin sites of healthy volunteers, with significantly lower quantification cycle (Cq) values observed in seborrheic dermatitis patients, suggesting an increased fungal burden. Burn patients also showed a marked increase in Malassezia spp. levels compared to healthy individuals. Stool samples demonstrated a low prevalence (6.7%) of Malassezia spp., but with high Cq values when present. Notably, ear samples revealed a higher positivity rate compared to nasal samples. The findings highlight the practicality and sensitivity of qPCR for elucidating the Malassezia burden across various human samples. This molecular approach confirms the differential colonization of Malassezia spp. in different clinical contexts. The panM-qPCR offers a promising approach for comprehensive mycobiota research, particularly in conditions where culture-based methods fall short.

Rapid Real-Time PCR Based on Core-Shell Tecto-Dendrimer-Entrapped Au Nanoparticles.

Rapid real-time PCR (generally <1 h) has broad prospects. In this study, we synthesized a new type of nanomaterial core-shell tecto-dendrimer coated with Au nanoparticles (Au CSTDs) for research in this field. The experimental results showed that Au CSTDs could significantly shorten the time of real-time PCR (from 72 to 28 min) with different templates, while the detection limit reached 10 copies and the nonspecific amplification was significantly reduced. Furthermore, experimental analyses and theoretical studies using the finite element simulation method confirmed that Au CSTDs function by synergistically enhancing electrostatic adsorption and thermal conductivity. These properties play a key role in improving real-time PCR, especially in particle-particle interactions. This study contributes an advanced method to rapid real-time PCR, which is expected to remarkably improve the efficiency, lower the detection limit, and enhance the specificity of molecular detection.

A systematic review on the selection of reference genes for gene expression studies in rodents: are the classics the best choice?

Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.

Diagnostic Accuracy of Five Molecular Assays for the Detection of Dengue Virus.

Background and Objectives: The steady spread of dengue virus (DENV) poses a profound public health threat worldwide. Reverse transcription real-time polymerase chain reaction (RT2-PCR) has been increasingly recognized as a reference method for the diagnosis of acute dengue infection. The goal of this study was to assess the diagnostic accuracy of five different RT2-PCR kits for the detection of DENV in a historically processed set of sera samples. Materials and Methods: In this retrospective study, 25 sera samples from routinely processed unique adult patients with a known DENV status (previously tested in both molecular and serological assays) were tested in parallel using four conventional (RealStar Dengue PCR Kit 3.0, Clonit'ngo Zika, Dengue & Chikungunya, BioPerfectus Zika Virus/Dengue Virus/Chikungunya Virus Real Time PCR Kit and Novaplex Tropical fever virus) and one sample-to-result (STANDARD M10 Arbovirus Panel) RT2-PCR assays. Additionally, an end-point dilution analysis was conducted in quintuplicate on six serial dilutions of an RNA preparation obtained from a culture-grown DENV serotype 1 strain for a total of 150 tests. Results: The overall accuracy of the evaluated tests ranged from 84% to 100%. In particular, the sensitivity of three conventional RT2-PCR assays (RealStar, Clonit'ngo and Novaplex) was 100% (95% CI: 79.6-100%), while it was lower (73.3%; 95% CI: 48.1-89.1%) for the BioPerfectus kit. The sample-to-result STANDARD M10 panel performed comparatively well, showing a sensitivity of 92.9% (95% CI: 68.5-98.7%). No false positive results were registered in any assay. The end-point dilution analysis suggested that the RealStar kit had the lowest limit of detection. Conclusions: Available RT2-PCR kits for the detection of DENV are highly specific and generally sensitive and, therefore, their implementation in diagnostic pathways is advisable.

A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA.

To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/µL), elevator buttons (11.9 pg/µL), cleaning supplies (7.17 pg/µL), and dispensers (16.4 pg/µL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10-8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations.