Simplex Assays Research Articles

Development of a triplex RT-qPCR assay for simultaneous quantification of Japanese encephalitis, Murray Valley encephalitis, and West Nile viruses for environmental surveillance.

The co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) has impacted human and animal health in multiple countries worldwide. To facilitate early warnings and surveillance of the presence of these viral infectious agents in the environment, a triplex reverse transcription-quantitative PCR (RT-qPCR) was developed for simultaneous quantification of JEV, MVEV, and WNV in potential hotspots such as piggery and urban wastewater and environmental water samples. The performance of the developed triplex RT-qPCR assay was compared with that of simplex counterparts, all using the same primer and probe sequences. The quantifiable results showed a concordance rate of 93.9%-100% (Cohen's kappa) between the triplex and simplex assays. The mean concentrations of exogenous JEV, MVEV, and WNV using the triplex and simplex RT-qPCR assays were remarkably similar in piggery/urban wastewater and environmental water samples. However, the impacts of the matrix effects (i.e., sample composition and PCR inhibition) of environmental water samples on the accurate quantification of these viruses need to be considered. Taken together, this newly developed triplex RT-qPCR assay of JEV, MVEV, and WNV will allow for a more rapid and cost-efficient sample analysis and data interpretation. The application of the triplex assay for environmental surveillance may be a valuable tool to complement the existing disease and mosquito surveillance approaches used to safeguard the health of both humans and animals.IMPORTANCEThe co-circulation of mosquito-borne Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and West Nile virus (WNV) poses significant threats to human and animal health globally. In this study, a triplex RT-qPCR assay was developed for simultaneous quantification of these viruses in wastewater and environmental water samples. Results demonstrated high concordance and sensitivity of the newly developed triplex RT-qPCR assay compared to simplex assays, indicating its efficacy for environmental surveillance. This cost-effective and rapid assay offers a vital tool for timely monitoring of mosquito-borne viruses in environmental samples, enhancing our ability to mitigate potential outbreaks and safeguard public health.

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL−1 bacterial DNA and 1 × 102 CFU mL−1 (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.

Development of Multiplex RT-PCR Assay for the Simultaneous Detection of Four Systemic Diseases Infecting Citrus

The citrus systemic diseases, including citrus Huanglongbing (caused by Candidatus Liberibacter asiaticus (CLas)), citrus tristeza (caused by citrus tristeza virus (CTV)), citrus tatter leaf (caused by citrus tatter leaf virus (CTLV)), and citrus exocortis (caused by citrus exocortis viroid (CEVd)), are threats to citrus production in Taiwan. Reliable diagnostic methods are important for the management of these systemic diseases. In this study, we developed a multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay to detect four pathogens simultaneously. Herein, the specific amplicons from each pathogen (295 bp for CLas, 468 bp for CTV, 120 bp for CTLV, and 196 bp for CEVd) were successfully produced using the optimized multiplex RT-PCR described here. The sensitivity evaluation showed that low titers of pathogens could be detected using this multiplex RT-PCR. Compared with the published simplex assays, the detection of field samples using the multiplex RT-PCR developed in this study showed a better performance. The detections using multiplex RT-PCR revealed that these four citrus systemic pathogens were commonly found in fields, and 30.0% of field samples were mix-infected. To our knowledge, this is the first study of a survey of the four important citrus systemic diseases in Taiwan, and it provides insights for improving disease management. Therefore, the multiplex RT-PCR assay provides a useful method for routine disease surveying and the production of pathogen-free citrus plants.

Differential detection of sheep and goat meat using duplex real-time PCR and high-resolution melt analysis

Authenticity in meat and meat products is of paramount importance in preventing adulteration and ensuring food safety. Since morphologically indistinct samples like sheep and goat meat requires accurate and sensitive differentiation, we developed and validated a duplex real-time polymerase chain reaction (qPCR) assay with high-resolution melt analysis (HRMA) for simultaneous identification of sheep and goat meat. Species-specific amplicons with melting temperature of 78 °C for goat and 80 °C for sheep (with associated standard errors) was identified. The assay was optimized for basic PCR parameters and the HRMA detection was standardized based on melt curve shape, color and inflexion points. The absolute limit of detection (LODabs) and limit of quantification (LOQ) of the assay were found to be 0.39 ng and 0.78 ng of target DNA respectively. A sensitivity of 0.1% was obtained for both sheep and goat meat. The relative limit of detection (LODrel) was also found to be 5%. The linearity and efficiency were evaluated in both simplex and duplex assays. The assay applicability was evaluated using proficiency test samples and commercially available raw and processed meat samples. The obtained results showed that this closed-tube qPCR-HRMA method can be a rapid and sensitive detection technique for meat speciation.

Development of a droplet digital PCR for pertussis toxin locus copy number determination in a genetically-modified Bordetella pertussis strain

To improve pertussis toxin (PT) yield in B. pertussis strains for vaccine production a genetically-engineered strain (gdPT 191-134 strain) with a second copy of the genetically detoxified PT (gdPT) locus was developed. The consistency of the production and genetic stability of the strain when used for vaccine production must be established. We developed two simplex ddPCR assays with PCR systems for ptxA, the target gene present in two copies, and pgm, the reference gene present as a single copy. The ddPCR assay had sufficient precision to discriminate the copy number of the PT locus accurately in two B. pertussis strains: one copy in the parent, non-genetically-engineered strain and two copies in the gdPT 191-134 strain. Using the ddPCR assays, we were able to show that the ratio of the ptxA to pgm genes decreased during serial culture passages, due to the loss of PT locus, which in turn, resulted in lower levels of PT production over time. We were then able to assess culture conditions that improved the stability of the double locus, as shown by non-significant reduction in gdPT toxin yield.

LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32°C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

Comparative Analysis of In-House RT-qPCR Detection of SARS-CoV-2 for Resource-Constrained Settings.

We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex assays adapted from the initial World Health Organization- (WHO) recommended protocol. This protocol included the identification of the E envelope gene (E gene; specific to the Sarvecovirus genus), RdRp gene of the RNA-dependent RNA polymerase (specific for SARS-CoV-2), and RNase P gene as endogenous control. The detection limit of the E and the RdRp genes were 3.8 copies and 33.8 copies per 1 µL of RNA, respectively, in both triplex and duplex reactions. The sensitivity for the RdRp gene in the triplex and duplex RT-qPCR tests were 98.3% and 83.1%, respectively. We showed a decrease in sensitivity for the RdRp gene by 60% when the E gene acquired Ct values > 31 in the diagnostic tests. This is associated with the specific detection limit of each gene and possible interferences in the protocol. Hence, developing efficient and cost-effective methodologies that can be adapted to various health emergency scenarios is important, especially in developing countries or settings where resources are limited.

Boosting SARS-CoV-2 detection combining pooling and multiplex strategies

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario.

A Robust and Fast/Multiplex Pharmacogenetics Assay to Simultaneously Analyze 17 Clinically Relevant Genetic Polymorphisms in CYP3A4, CYP3A5, CYP1A2, CYP2C9, CYP2C19, CYP2D6, ABCB1, and VKORC1 Genes.

In the field of pharmacogenetics, the trend is to analyze a panel of several actionable genetic polymorphisms. It may require the use of high-throughput sequencing which demands expensive reagents/instruments and specific skills to interpret results. As an alternative, the aim of this work was to validate an easy, fast, and inexpensive multiplex pharmacogenetics assay to simultaneously genotype a panel of 17 clinically actionable variants involved in drug pharmacokinetics/pharmacodynamics. We designed primers to perform a multiplex PCR assay using a single mix. Primers were labeled by two fluorescent dye markers to discriminate alleles, while the size of the PCR fragments analyzed by electrophoresis allowed identifying amplicon. Polymorphisms of interest were CYP3A4*22, CYP3A5*3, CYP1A2*1F, CYP2C9*2-*3, CYP2C19*2-*3-*17, VKORC1-1639G > A, ABCB1 rs1045642-rs1128503-rs2229109-rs2032582, and CYP2D6*3-*4-*6-*9. The assay was repeatable and a minimum quantity of 10 ng of DNA/ sample was needed to obtain accurate results. The method was applied to a validation cohort of 121 samples and genotyping results were consistent with those obtained with reference methods. The assay was fast and cost-effective with results being available within one working-day. This robust assay can easily be implemented in laboratories as an alternative to cumbersome simplex assays or expensive multiplex approaches. Together it should widespread access to pharmacogenetics in clinical routine practice.

One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

Droplet digital polymerase chain reaction (ddPCR) is a third generation of PCR that was recently developed to overcome the limitation of direct quantification observed in real-time quantification PCR (qPCR). Recent studies have shown that ddPCR is more sensitive than the gold standard reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. In combination with multiplexing, multiple RT-ddPCR assays can be developed to directly quantify different SARS-CoV-2 nucleic acid targets within a single sample, significantly saving on cost and time. Since ddPCR is tolerant to a number of inhibitors unlike qPCR, it can be used to detect and quantify samples from complex environments like wastewater. Here we present three one-step RT-ddPCR protocols on how to develop simplex (one target), duplex (two targets), and triplex probe mix (three targets) assays for SARS-CoV-2 detection and quantification. The assays can be used for diagnosis or other research-related SARS-CoV-2 applications.

Development of simplex and multiplex RT-qPCR assays for the detection of three cryptic viruses of black-grass (Alopecurus myosuroides)

Simplex and multiplex RT-qPCR assays were developed for Alopecurus myosuroides partitivirus 1 (AMPV1), Alopecurus myosuroides partitivirus 2 (AMPV2) and Alopecurus myosuroides varicosavirus 1 (AMVV1), and compared to the existing conventional PCR assays. All assays had a high specificity and their sensitivity was increased compared to the conventional RT-PCR assays. As viral quantification is an important element in comparative experiments, the effect of high- and low-temperature drying treatments, prior to RNA extraction and analysis, was studied and optimised. AMVV1 detection was reduced by both drying treatments, but particularly by the high-temperature. AMPV1 and AMPV2 detection on the other hand was not impeded by the drying treatments, and enables standardisation of plant tissue prior to extraction, in particular for quantitative analysis.

Evaluation of serological assays to monitor antibody responses to single-dose HPV vaccines

IntroductionWhether existing serological assays are sufficiently robust to measure the lower antibody levels expected following single-dose HPV vaccination is unknown. MethodsWe evaluated seven assays measuring HPV-16/18 immunological responses overall and by number of doses in 530 serum samples from participants receiving varying doses of Cervarix or Gardasil up to 36-months post-vaccination. Serum was evaluated by simplex (HPV-16 ELISA, HPV-18 ELISA), multiplex (LIA-4, VLP-MIA, M9ELISA, GST-L1), and high-throughput pseudovirion-based neutralization assays (HT-PBNA), and results were compared to the gold standard HPV-16/18 secreted alkaline phosphatase neutralization assay (SEAP-NA). Reproducibility was assessed by the coefficient of variation (CV) and intraclass correlation coefficient (ICC). Percent agreement, Pearson correlation, and weighted-kappa were used to assess validity. Determinants of seronegativity were evaluated by chi-squared test. ResultsHPV-16: Seropositivity range was 97.1–99.5% for single dose and 98.8–99.8% overall. CV range was 4.0–18.0% for single dose and 2.9–19.5% overall. ICC range was 0.77–0.99 for single dose and 0.74–0.99 overall. Correlation with SEAP-NA range was 0.43–0.85 for single dose and 0.51–0.90 overall. Weighted-kappa range was 0.34–0.82 for single dose and 0.45–0.84 overall. HPV-18: Seropositivity range was 63.9–94.7% for single dose and 86.2–97.9% overall. CV range was 8.1–18.2% for single dose and 4.6–18.6% overall. ICC range was 0.75–0.99 for single dose and 0.83–0.99 overall. Correlation with SEAP-NA range was 0.31–0.99 for single dose and 0.27–0.96 overall. Weighted-kappa range was 0.35–0.83 for single dose and 0.45–0.84 overall. HPV-16 seronegativity was <5% for all assays. HPV-18 seronegativity range was 5.5–17.3%. For LIA-4 and GST-L1 where the proportion of seronegativity was >10%, the strongest correlates of seronegativity were receiving a single vaccine dose and receiving Gardasil. Conclusions:These results support the utility of existing serological assays to monitor antibody responses following single-dose HPV vaccination.

Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of Mycobacterium tuberculosis.

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works.

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues.

Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 103 cells.ml−1. Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples.

A duplex PCR assay for the simultaneous quantification of Bacteroides HF183 and crAssphage CPQ_056 marker genes in untreated sewage and stormwater

The HF183 marker gene, derived from the 16S rRNA gene of Bacteroides dorei, has been widely used to identify sewage pollution in environmental waters. CrAssphages are recently discovered DNA bacteriophages that are highly abundant in untreated sewage and have shown promises for tracking sewage contamination in environmental waters. In this paper, we report the development of a duplex quantitative PCR (qPCR) assay for simultaneous quantification of HF183 and crAssphage CPQ_056 marker genes in untreated sewage and sewage impacted stormwater. Same primer and probe sequences were used in the duplex qPCR assay as used in published simplex qPCR assays. The performance characteristics of the duplex qPCR assay were similar to its simplex counterparts. We validated the performance of the duplex assay in a collaborative laboratory study with the aim to evaluate reproducibility, sensitivity and concordance for field study. The concordance values between the simplex vs. duplex qPCR assays for HF183 and crAssphage CPQ_056 marker genes ranged from 96.7 to 100% and the mean concentrations of HF183 and CPQ_056 in environmental water samples were remarkably similar or in some cases slightly greater for the duplex qPCR assay suggesting the reliability of this assay for monitoring HF183 and CPQ_056 simultaneously. The newly developed duplex qPCR assay will be a valuable addition to the MST toolbox for sewage pollution monitoring and would allow rapid and comparative sample analysis.

Development and Evaluation of a Triplex TaqMan Assay and Next-Generation Sequence Analysis for Improved Detection of Xylella in Plant Material.

Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but it causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica, and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools is indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next-generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain, and Italy, or intercepted in Austria and the Netherlands, were analyzed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified to the subspecies level, and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS-analyzed samples were confirmed with the triplex TaqMan polymerase chain reaction and loop-mediated isothermal amplification.

Development of real-time PCR assays for single and simultaneous detection of infectious bursal disease virus and chicken anemia virus

Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) cause relevant immunosuppressive diseases in poultry. Clinical diagnosis of these viruses is challenging given the different disease presentations and the frequent occurrence of co-infections with other pathogens. Here, we standardized and validated simplex and duplex RT-qPCR assays for the straightforward detection of IBDV and CAV. The qPCR assays are based on primers and hydrolysis probes that target highly conserved regions of IBDV and CAV genomes. Analytical sensitivity tests on 10-fold serial dilutions containing 100-108 viral genomes indicated that the simplex assays have good determination coefficients and efficiency and detect a wide range of virus doses (102 to 108 molecules copies/reactions). The relatively small values of intra- and inter-assay variability ensure the repeatability and support its reproducibility in different diagnostic and research facilities. The assays are also efficient tools for absolute quantification as indicated by the analytical performance analysis. The assays have an excellent specificity and absence of cross-reactivity with negative samples, or with other common avian viruses. The simplex IBDV and CAV assays use probes labelled with different dyes (FAM and HEX) and can be multiplexed for the simultaneous detection of both viruses. The determination coefficients, PCR efficiencies, and relatively small intra- and inter-assay variability were comparable to the simplex assays. This duplex assay is the first to simultaneously detect IBDV and CAV using the same RNA extraction from the bursa of Fabricius in a single and straightforward step. Therefore, this method is time saving, provides quantitative results for both targets without any cross-reaction, and reduces the risk of carrying-over contaminations. The qPCR assays here developed can be used in simplex and duplex formats for detection and quantification of large number of samples with reliable sensitivity and specificity. These tools are expected to improve surveillance and control of these ubiquitous viruses.

Microlens Enhancement of Surface-Tethered Molecular Beacons.

The tethering of molecular beacon oligonucleotide detection probes to surface-patterned poly(ethylene glycol) (PEG) microgels has enabled the integration of molecular beacons into a microarray format. The microgels not only localize the probes to specific surface positions but also maintain them in a waterlike environment. Here we extend the concept of microgel tethering to include dielectric microlenses. We show that streptavidin-functionalized polystyrene microspheres (3 μm diameter) can be colocalized with molecular beacons using biotinylated PEG gels in patterns ranging from pseudocontinuous microgel pads with lateral dimensions on the order of tens of micrometers to individual microgels with lateral dimensions on the order of 400-500 nm. We use a simplex assay based on Influenza A detection to study the lensing behavior. The microspheres increase the effective numerical aperture of the collection optics, and we find that a tethered microsphere increases the peak intensity collected from hybridized beacons between 1.5 and 10 times depending on the specific pattern size and areal density of microgels. The highest signal increase occurs when a single microsphere is tethered to a single isolated microgel. The tethering is highly self-directed and occurs in the individual-microgel case only when the microgel is close to the optic axis of the microsphere. This alignment minimizes spherical aberration and maximizes coupling of emitted fluorescent intensity into the collection optics.

SensiScreen®KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations.

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase’s proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25–1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods.

A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification.

The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarker. There is much evidence indicating that a panel of miRNAs, termed “miRNA fingerprints”, is more specific and informative than an individual miRNA as biomarker. Thus, multiplex assays for simultaneous quantification of multiple miRNAs could be more potent in clinical practice. However, current available assays normally require pre-enrichment, amplification and labeling steps, and most of them are semi-quantitative or lack of multiplexing capability. In this study, we developed a quasi-targeted proteomics assay for multiplexed miRNA quantification by a combination of DNA-peptide probes and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, the signal of target miRNAs (i.e., miR-21, miR-let7a, miR-200c, miR-125a and miR-15b) was converted into the mass response of reporter peptides by hybridization of miRNAs with DNA-peptide probes and subsequent tryptic digestion to release the peptides. After a careful optimization of conditions related to binding, conjugation, hybridization and multiple reaction monitoring (MRM) detection, the assay was validated for each miRNA and the limit of quantification (LOQ) for all the miRNAs can achieve 1 pM. Moreover, crosstalk between DNA-peptide probes in multiplex assay was sophisticatedly evaluated. Using this quasi-targeted proteomics assay, the level of target miRNAs was determined in 3 human breast cell lines and 36 matched pairs of breast tissue samples. Finally, simplex assay and qRT-PCR were also performed for a comparison. This approach grafts the strategy of targeted proteomics into miRNA quantification and may offer a new way for multiplexed miRNA profiling.