Probe-based qPCR Research Articles

TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines.

Background: There has been global concern about the safety and accuracy of traditional Chinese patent medicines (TCPMs). Panax notoginseng, also known as sanqi, is an important constituent of TCPMs. However, identifying the species contained in TCPMs is challenging due to the presence of multiple ingredients and the use of various preparation processes. Objective: To detect P. notoginseng in TCPMs. Methods: A TaqMan probe-based qPCR assay was constructed and validated with DNA extracted from P. notoginseng and adulterants. In total, 75 samples derived from 25 batches of TCPMs were tested using the constructed qPCR method. Results: A TaqMan probe-based qPCR assay targeting P. notoginseng was established. The constructed qPCR assay could specifically discriminate P. notoginseng from Panax ginseng, Panax quinquefolium and Curcuma aromatica Salisb. cv. Wenyujin. The sensitivity study showed that the detectable DNA template concentration of P. notoginseng for this qPCR assay was 0.001 ng/μl. All 75 samples from TCPMs were confirmed to contain P. notoginseng by the qPCR assay. Conclusions: The qPCR method can accurately identify P. notoginseng in TCPMs and is promising as a powerful tool for quality control and market regulation.

Sprouting Angiogenesis in Human Pituitary Adenomas.

IntroductionAngiogenesis in pituitary tumors is not fully understood, and a better understanding could help inform new pharmacologic therapies, particularly for aggressive pituitary tumors.Materials and Methods219 human pituitary tumors and 12 normal pituitary glands were studied. Angiogenic genes were quantified by an angiogenesis qPCR array and a TaqMan probe-based absolute qPCR. Angiogenesis inhibition in pituitary tumors was evaluated in vitro with the endothelial tube formation assay and in vivo in RbΔ19 mice.Results71 angiogenic genes, 40 of which are known to be involved in sprouting angiogenesis, were differentially expressed in pituitary tumors. Expression of endothelial markers CD31, CD34, and ENG was significantly higher in pituitary tumors, by 5.6, 22.3, and 8.2-fold, respectively, compared to in normal pituitary tissue. There was no significant difference in levels of the lymphatic endothelial marker LYVE1 in pituitary tumors compared with normal pituitary gland tissue. Pituitary tumors also expressed significantly higher levels of angiogenesis growth factors, including VEGFA (4.2-fold), VEGFB (2.2), VEGFC (19.3), PGF (13.4), ANGPT2 (9.2), PDGFA (2.7), PDGFB (10.5) and TGFB1 (3.8) compared to normal pituitary tissue. Expression of VEGFC and PGF was highly correlated with the expression of endothelial markers in tumor samples, including CD31, CD34, and ENG (endoglin, a co-receptor for TGFβ). Furthermore, VEGFR inhibitors inhibited angiogenesis induced by human pituitary tumors and prolonged survival of RbΔ19 mice.ConclusionHuman pituitary tumors are characterized by more active angiogenesis than normal pituitary gland tissue in a manner consistent with sprouting angiogenesis. Angiogenesis in pituitary tumors is regulated mainly by PGF and VEGFC, not VEGFA and VEGFB. Angiogenesis inhibitors, such as the VEGFR2 inhibitor cabozantinib, may merit further investigation as therapies for aggressive human pituitary tumors.

Detection and population genomics of sea turtle species via noninvasive environmental DNA analysis of nesting beach sand tracks and oceanic water.

Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA - the detection of DNA shed from organisms into their surrounding environments. We developed species-specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe-based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in-water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that noninvasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g., biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles.

Probe-based qPCR assay enables the rapid and specific detection of bacterial degrading genes for the pesticide metaldehyde in soil

Metaldehyde, a molluscicide pesticide, has been identified as a pollutant of concern due to its repeated detection in drinking water, thereby generating numerous compliance failures for water utilities. Biological degradation potential for metaldehyde is widespread in soils, occurring at different rates, but to date, no molecular methods for its assessment have been reported. Here, three genes belonging to a shared metaldehyde-degrading gene cluster present in bacteria were used as candidates for development of a quantitative PCR (qPCR) assay for assessing the metaldehyde-degrading potential in soil. Screening of gene targets, primer pairs and optimization of reaction conditions led to the development of a sensitive and specific probe-based qPCR method for quantifying the mahY metaldehyde-degrading gene from soil. The technique was tested across 8 soils with different compositions and origins. The degrading pathway was detected in 4/8 soils, in which a higher number of gene copies correlated with periods of greater metaldehyde removal. Additionally, swift elimination of the pesticide was observed in soils with an elevated initial number of mahY gene copies. The gene cluster was not detected in other soils, even though metaldehyde removal occurred, indicating that other biological degrading pathways are also important in nature. The method described here is the first one available to estimate the microbial metaldehyde degradation potential and activity in soils, and can also be used to detect degrading microorganisms in systems such as sand filters for water purification or to monitor degrading strains in engineered processes.

Worldwide distribution and environmental origin of the Adelaide imipenemase (AIM-1), a potent carbapenemase in Pseudomonas aeruginosa.

Carbapenems are potent broad-spectrum β-lactam antibiotics reserved for the treatment of serious infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa . The surge in P. aeruginosa resistant to carbapenems is an urgent threat, as very few treatment options remain. Resistance to carbapenems is predominantly due to the presence of carbapenemase enzymes. The assessment of 147 P . aeruginosa isolates revealed that 32 isolates were carbapenem non-wild-type. These isolates were screened for carbapenem resistance genes using PCR. One isolate from wastewater contained the Adelaide imipenemase gene (bla AIM-1) and was compared phenotypically with a highly carbapenem-resistant clinical isolate containing the bla AIM-1 gene. A further investigation of wastewater samples from various local healthcare and non-healthcare sources as well as river water, using probe-based qPCR, revealed the presence of the bla AIM-1 gene in all the samples analysed. The widespread occurrence of bla AIM-1 throughout Adelaide hinted at the possibility of more generally extensive spread of this gene than originally thought. A blast search revealed the presence of the bla AIM-1 gene in Asia, North America and Europe. To elucidate the identity of the organism(s) carrying the bla AIM-1 gene, shotgun metagenomic sequencing was conducted on three wastewater samples from different locations. Comparison of these nucleotide sequences with a whole-genome sequence of a P. aeruginosa isolate revealed that, unlike the genetic environment and arrangement in P. aeruginosa , the bla AIM-1 gene was not carried as part of any mobile genetic elements. A phylogenetic tree constructed with the deduced amino acid sequences of AIM-1 suggested that the potential origin of the bla AIM-1 gene in P. aeruginosa might be the non-pathogenic environmental organism, Pseudoxanthomonas mexicana .

Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping

BackgroundThe detection and identification of single nucleotide polymorphism (SNP) is essential for determining patient disease susceptibility and the delivery of medicines targeted to the individual. At present, SNP genotyping technology includes Sanger sequencing, TaqMan-probe quantitative polymerase chain reaction (qPCR), amplification-refractory mutation system (ARMS)-PCR, and Kompetitive Allele-Specific PCR (KASP). However, these technologies have some disadvantages: the high cost of development and detection, long and time consuming protocols, and high false positive rates. Focusing on these limitations, we proposed a new SNP detection method named universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR). In this method, only two types of fluorescence-labeled probes were used for SNP genotyping, thus greatly reducing the cost of development and detection for SNP genotyping.ResultsIn the amplification process of UPIP-qPCR, unlabeled intermediate primers with template-specific recognition functions could trigger probe hydrolysis and specific signal release. UPIP-qPCR can be used successfully and widely for SNP genotyping. The sensitivity of UPIP-qPCR in SNP genotyping was 0.01 ng, the call rate was more than 99.1%, and the accuracy was more than 99.9%. High-throughput DNA microarrays based on intermediate primers can be used for SNP genotyping.ConclusionThis novel approach is both cost effective and highly accurate; it is a reliable SNP genotyping method that would serve the needs of the clinician in the provision of targeted medicine.

Development of Hydrolysis Probe-Based qPCR Assays for Panax ginseng and Panax quinquefolius for Detection of Adulteration in Ginseng Herbal Products.

Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species; (2) sensitivity, in detecting the lowest amount of the target material; and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80–120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs; authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis.

Sporothrix schenckii and related species are the agents of human and animal sporotrichosis. Routine diagnoses using classical mycological approaches are unspecific due to overlapping phenotypes. As the frequency and prevalence of sporotrichosis increases worldwide, developing specific, sensitive and cost-effective diagnostic tools is essential to understand the distribution patterns, map-affected areas and promote specific public health strategies to mitigate future outbreaks. Polymorphisms among the β-tubulin gene were exploited to speciate S. brasiliensis, S. schenckii and S. globosa in a one-tube multiplex probe-based qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC=1.000, 95% CI=0.971-1.000, p<.0001) without cross-reacting with other medically relevant fungi, human, feline or murine DNA. Speciation via multiplex qPCR matched phylogenetic identification (Kappa=1.0; 95% CI=1.0-1.0; very good agreement), supporting its use as a reliable alternative to DNA sequencing. Remarkably, the lower limit of detection was 3 copies of the target for all species. As a proof of concept, we used swabs of wound exudate of 70 cats suspected of sporotrichosis to reveal an overwhelming occurrence of S. brasiliensis in 69 specimens (sensitivity=98.57%; 95%CI: 92.3-100.0 and specificity=100%; 95% CI=78.2-100). In comparison to culture, qPCR showed a larger area under the curve (AUC=0.993±0.007; 95% CI=0.944-1.000; p<.0001; Youden's index=0.9857), supporting that qPCR is an essential tool for accurately detect Sporothrix DNA directly from clinical samples, thus accelerating the diagnosis of sporotrichosis. Moreover, our multiplex qPCR system has the potential to increase diagnostic capacity in Sporothrix-affected areas, helping the local animal health agent or veterinarian to quickly identify and isolate new cases, which will likely benefit thousands of patients infected every year worldwide.

Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow.

Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel.

Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

Mitochondrial DNA 4977 bp Deletion in Peripheral Blood Is Associated With Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder worldwide. We aimed to examine the associations of two mitochondrial DNA (mtDNA) biomarkers in the peripheral blood, mtDNA copy number (CN), and mtDNA4977 deletion rate (DR), with PCOS in a clinical setting.MethodsWe performed a study involving 263 women with PCOS and 326 age-matched controls between June 2015 and June 2019. The mtDNA CN and mtDNA4977 DR were measured using multiplex probe-based qPCR. The associations of the mtDNA CN and mtDNA4977 DR with the risk of PCOS were estimated using logistic regression.ResultsAnalysis of the associations between mtDNA biomarkers and PCOS indicate that the mtDNA CN (P = 0.003) and mtDNA4977 DR (P < 0.001) in PCOS patients were significantly higher than those in the controls. After adjusting for the body mass index, luteinizing hormone/follicle-stimulating hormone ratio, and testosterone level, only higher mtDNA4977 DR was associated with PCOS (odds ratio 1.053, 95% confidence interval 1.024 to 1.083; P < 0.001). The linear dose-response trends of the mtDNA4977 DR were also supported by the quartile analysis.ConclusionMultivariable models suggest that mtDNA4977 DR levels are strongly associated with PCOS and represent an independent risk factor for PCOS. Further investigation of the utility of mtDNA as a biomarker for PCOS is warranted.

Preliminary analysis of whether mosquitoes can carry and transmit African swine fever

BackgroundMosquitoes are important insect vectors, but whether they can carry and transmit African swine fever virus (ASFV) in large-scale pig farms in China is unknown.ResultsIn this study, probe-based qPCR analysis was performed on mosquitoes from five pig farms with ASF virus (ASFV). Analysis of ASFV in 463 mosquitoes yielded negative cycle threshold (CT) value), and detection remained negative after mixing samples from all five pig farms.ConclusionsTherefore, mosquitoes appear unlikely to transmit ASFV, and pose little threat to large-scale pig farms. Thus, farms should continue to follow normal mosquito control procedures when formulating strategies for the prevention and control of ASF.

Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species.

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.

Next generation sequencing reveals limitation of qPCR methods in quantifying emerging antibiotic resistance genes (ARGs) in the environment.

Occurrence of emerging antibiotic resistance genes (ARGs) in the environment, especially those conferring resistance to the last resort antibiotic carbapenems (blaKPC) and colistin (mcr-1), has become an important environmental issue. Real-time polymerase chain reaction (qPCR) methods were commonly used to quantify emerging ARGs in the environment, with some studies reporting high abundance. Due to the high diversity of DNA templates and complexity in environmental samples, overestimation or even false-positive detection of target genes may occur due to potential non-specific amplification. This study compared the performance of dye-based qPCR and probe-based qPCR assays for the detection of blaKPC and mcr-1 in activated sludge (AS) samples, which showed that the detection of blaKPC and mcr-1 by the dye-based qPCR assays was likely false-positive when compared with probe-based qPCR results. Next generation sequencing (NGS) of the qPCR reactions identified primer dimers and non-specific amplicons as the primary causes for the false-positive detection. NGS also detected target amplicons in the negative reactions, indicating potential false-negative detection by the probe-based qPCR assays. Testing of spiked samples showed false-positive detection and overestimation by the dye-based qPCR assays primarily occurred at low concentrations of target DNA, while false-negative detection by probe-based qPCR was caused primarily by reduced amplification efficiencies in the environmental samples. Together, the results identified the limitations of the qPCR methods in complex environmental samples and demonstrated the remedial utility of NGS in quantifying emerging ARGs by qPCR. KEY POINTS: • Dye-based qPCR assays are prone to false-positive and overestimation at low substrate concentration. • Probe-based qPCR assays can experience false-negative detection due to environmental interference. • The combination of qPCR with NGS provides improved sensitivity and reliability in detecting low level ARGs in complex environmental matrices.

The Concurrent Detection of Chelonid Alphaherpesvirus 5 and Chelonia mydas Papillomavirus 1 in Tumoured and Non-Tumoured Green Turtles.

Simple SummaryCharacterised by benign tumours, fibropapillomatosis is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of evidence has associated these tumours with a herpesvirus. However, a recent study detected both herpesvirus and papillomavirus in these tumours. This result challenged the idea that the herpesvirus is the sole virus associated with this disease. The present study aimed to better understand the co-occurrence of these viruses in turtles with fibropapillomatosis (in both tumour samples and non-tumoured skin samples), in addition to samples from non-tumoured turtles. Both viruses were detected in all sample types, with the 43.5% of tumours containing both herpesvirus and papillomavirus. Tumour samples were found to contain the most herpesvirus while the highest amount of papillomavirus was detected in non-tumoured skin from turtles with tumours. Collectively, these results pivot the way we think about this disease; as an infectious disease where two separate viruses may be at play.Characterised by benign tumours, fibropapillomatosis (FP) is a debilitating disease that predominantly afflicts the endangered green turtle (Chelonia mydas). A growing body of histological and molecular evidence has associated FP tumours with Chelonid alphaherpesvirus 5 (ChHV5). However, a recent study which detected both ChHV5 and Chelonia mydas papillomavirus 1 (CmPV1) DNA in FP tumour tissues has challenged this hypothesis. The present study aimed to establish a probe-based qPCR to assess the wider prevalence of CmPV1 and co-occurrence with ChHV5 in 275 marine turtles foraging in waters adjacent to the east coast of Queensland, Australia: three categories: Group A (FP tumours), Group B (non-tumoured skin from FP turtles) and Group C (non-tumoured skin from turtles without FP). Concurrent detection of ChHV5 and CmPV1 DNA is reported for all three categories, where Group A had the highest rate (43.5%). ChHV5 viral loads in Group A were significantly higher than loads seen in Group B and C. This was not the case for CmPV1 where the loads in Group B were highest, followed by Group A. However, the mean CmPV1 load for Group A samples was not significantly different to the mean load reported from Group B or C samples. Collectively, these results pivot the way we think about FP; as an infectious disease where two separate viruses may be at play.

PANDAA intentionally violates conventional qPCR design to enable durable, mismatch-agnostic detection of highly polymorphic pathogens

Sensitive and reproducible diagnostics are fundamental to containing the spread of existing and emerging pathogens. Despite the reliance of clinical virology on qPCR, technical challenges persist that compromise their reliability for sustainable epidemic containment as sequence instability in probe-binding regions produces false-negative results. We systematically violated canonical qPCR design principles to develop a Pan-Degenerate Amplification and Adaptation (PANDAA), a point mutation assay that mitigates the impact of sequence variation on probe-based qPCR performance. Using HIV-1 as a model system, we optimized and validated PANDAA to detect HIV drug resistance mutations (DRMs). Ultra-degenerate primers with 3’ termini overlapping the probe-binding site adapt the target through site-directed mutagenesis during qPCR to replace DRM-proximal sequence variation. PANDAA-quantified DRMs present at frequency ≥5% (2 h from nucleic acid to result) with a sensitivity and specificity of 96.9% and 97.5%, respectively. PANDAA is an innovative advancement with applicability to any pathogen where target-proximal genetic variability hinders diagnostic development.

The Epidome - a species-specific approach to assess the population structure and heterogeneity of Staphylococcus epidermidis colonization and infection

BackgroundAlthough generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunocompromized patients. Infections are often caused by multidrug-resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients’ quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive.In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities.ResultsBased on an assessment of > 800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. The approach was validated on isolates representing skin commensals and on genomic mock communities with a sensitivity of < 10 copies/μL. The method was furthermore applied to a sample set of primary skin and nasal samples, revealing a high degree of heterogeneity in the S. epidermidis populations. Additionally, the qPCR showed a high degree of variation in absolute abundance of S. epidermidis.ConclusionsThe Epidome method is designed for use on primary samples to obtain important information on S. epidermidis abundance and diversity beyond species-level to answer questions regarding the emergence and dissemination of nosocomial lineages, investigating clonality of S. epidermidis communities, population dynamics, and niche selection. Our targeted-sequencing method allows rapid differentiation and identification of clinically important nosocomial lineages in low-biomass samples such as skin samples.

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Fusobacterium nucleatum is associated with worse prognosis in Lauren\u2019s diffuse type gastric cancer patients

Fusobacterium nucleatum (F. nucleatum) is frequently detected in primary colorectal cancer (CRC) and matching metastasis, and has been linked to a worse prognosis. We investigated the presence of F. nucleatum in gastric cancer (GC) and gastric preneoplastic conditions of the stomach, and its potential prognostic value in GC patients. Fusobacterium spp. and F. nucleatum were quantified in various specimens from gastrointestinal tract including paired CRC and GC tissues using probe-based qPCR. Fusobacterium spp. and F. nucleatum were more frequently found in tumorous tissue of CRC and GC compared to non-tumorous tissues. The frequency and bacterial load were higher in CRC compared to GC patients. F. nucleatum positivity showed no association to chronic gastritis or preneoplastic conditions such as intestinal metaplasia. F. nucleatum-positivity was associated with significantly worse overall survival in patients with Lauren’s diffuse type, but not with intestinal type GC. There was no association with gender, Helicobacter pylori-status, tumor stage or tumor localization. However, F. nucleatum was positively associated with patient’s age and a trend for a lower global long interspersed element-1 DNA methylation. In conclusion, our work provides novel evidence for clinical relevance of F. nucleatum in GC by showing an association between F. nucleatum positivity with worse prognosis of patients with Laurens’s diffuse type gastric cancer. Further studies are necessary to explore related mechanistic insights and potential therapeutic benefit of targeted antibiotic treatment in GC patients.

Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula.

Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.