Duplex Assay Research Articles

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.

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/μL, 30.6 copies/μL and 60 copies/μL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

VGLUT-VGAT expression delineates functionally specialised populations of vasopressin-containing neurones including a glutamatergic perforant path-projecting cell group to the hippocampus in rat and mouse brain.

The origin and functional significance of vasopressin (AVP)-containing fibres in limbic regions has been an ongoing subject of investigation for several years. We have previously identified AVP-magnocellular neurones of rat hypothalamus that provide glutamatergic projections to the hippocampus, amygdala, lateral habenula and locus coeruleus. However, we also reported AVP-immunopositive fibres in those regions that are thin and make Gray type II synapses, which are unlikely to be of magnocellular origin. Therefore, in the present study, we characterised AVP mRNA co-expression with expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) neuronal traits in rat and mouse brain, using high-resolution in situ hybridisation methods, including a radio-ribonucleotide and RNAscope 2.5 HD duplex assay, with Slc17a7, Slc17a6, Slc32a1 and Avp probes corresponding to mRNAs of VGLUT1, VGLUT2, VGAT and AVP, respectively. We located 18 cell groups expressing Avpand identified their molecular signatures for VGLUT and VGAT mRNA expression. Avpcell groups of hypothalamus and midbrain are mainly VGLUT mRNA-expressing, whereas those in regions derived from cerebral nuclei are mainly VGAT mRNA-expressing, suggesting a functional segregation of glutamate/GABA co-transmission with AVP. A newly identified Slc17a7 and Slc17a6 (but not Slc32a1) expressing vasopressinergic cell group was found in layer II-III neurones of the central entorhinal cortex, which projects to the hippocampus. These data support the notion of a complex role for AVP with respect to modulating multiple central circuits controlling behaviour in specific ways depending on co-transmission with glutamate or GABA, potentially giving rise to a functional classification of AVPergic neurones in the central nervous system.

Serum estradiol levels in infertile men with non-obstructive azoospermia.

Purpose:To report the different patterns of estradiol levels in infertile men with non-obstructive azoospermia and correlate these levels with their clinical and laboratory findings.Materials and methods:A retrospective study was launched, and a retrieval of data for infertile men with non-obstructive azoospermia (n = 166) and fertile controls (n = 40) was performed. The retrieved data included demographics, clinical findings, scrotal duplex, semen analysis, and hormonal assay (testosterone, follicle-stimulating hormone, luteinizing hormone, prolactin, and estradiol).Results:Our findings showed a wide spectrum of estradiol concentrations. The patients were arranged into three groups (high, normal, and low estradiol groups). The normal estradiol group was the most prevalent (71.1%). Testosterone, gonadotrophins, testicular volumes, and the number of patients with jobs in polluted workplaces showed significant differences among the study groups (p = 0.001, <0.001, <0.001, and 0.004, respectively). Age, body mass index, varicocele prevalence, prolactin, and smoking habits did not show any significant differences among the groups. Obesity was lacking in the low estradiol group, but it had significantly higher prevalence in the normal (p = 0.013) or high group (p = 0.023) compared with the controls.Conclusion:Serum estradiol, in infertile men with non-obstructive azoospermia, may be present at different levels. It is recommended that estradiol be measured in infertile men with non-obstructive azoospermia when there is an alteration in testosterone concentration, obesity, a polluted workplace occupation, or before trying hormonal therapy. Extended studies are highly recommended to provide a clear clue whether alterations in estradiol concentrations in men with non-obstructive azoospermia are the cause or a consequence of the condition.

Semi-Quantitative, Duplexed qPCR Assay for the Detection of Leishmania spp. Using Bisulphite Conversion Technology

Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.

A New Protocol for Molecular Detection of Cyclospora cayetanensis as Contaminants of Berry Fruits.

Cyclospora cayetanensis is a coccidian parasite that is associated with foodborne outbreaks of gastrointestinal illnesses. Raspberries have been implicated as a vehicle of infection in some of these outbreaks. Most of the molecular techniques used for the detection of parasites commonly use the 18s rRNA as a target gene, which is highly conserved. The conserved nature of the 18s rRNA gene among coccidia means that there is potential for cross-reactivity from primers intended to target this gene in C. cayetanensis with the same gene in related coccidia. This provides an additional challenge in developing a specific detection method. The aim of this study is to develop a new, more specific assay to detect C. cayetanensis in berry fruits. This new assay, targeting the internal transcribed spacer 1 (ITS-1) region, was tested on three different berry matrices: raspberries, blueberries, and strawberries. The new assay showed good efficiency (102%), linearity (r2 = 0.999), repeatability (standard deviation of Cq 0.2 (95% CI: 0.2, 0.3) and specificity for Cyclospora, with no cross-reactivity with related coccidia (Toxoplasma gondii, Eimeria mitis, Cystoisospora canis, and Cryptosporidium parvum) when tested in vitro. The method development was initially conducted using Cyclospora DNA only. After it was confirmed to have an acceptable performance, the method was evaluated using the oocysts of C. cayetanensis. The method was also improved by incorporating an internal control as a duplex in order to monitor PCR inhibition due to sample matrix components. The duplex assay also showed a good efficiency (100%) and linearity (r2 = 0.99). The results showed that the new assay has potential for standard use in food testing laboratories. Furthermore, results regarding important factors related to assay robustness are discussed.

Abstract 4965: Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer

Abstract Immunotherapy has proven to be a powerful anti-tumor therapy, harnessing the body’s own immune system to target and kill tumor cells. However, immunotherapy is not successful in all cancer patients due to both intrinsic non-responsiveness and adaptive resistance. Developing predictive biomarkers and understanding mechanisms of resistance are major goals of the immuno-oncology community. The extracellular matrix (ECM), an important factor for promoting tumor growth, survival, and migration of tumor cells, can also act as a physical barrier to prevent immune cell infiltration and promote tumor immune escape. Components of the ECM such as COL11A1, COL4A1, and LOXL2 have been shown to be associated with cancer progression. Furthermore, new data suggests that TGFβ activation leads to up-regulation of ECM genes in cancer-associated fibroblasts and immune suppression. However, it remains poorly understood which cells in the tumor microenvironment (TME) are the sources of ECM gene expression and how they are related to tumor infiltrating cytotoxic T lymphocytes (CTLs). In this study, we employed a highly sensitive and specific RNAscope in situ hybridization (ISH) duplex assay to directly visualize the tissue distribution of cells expressing COL4A1, COL11A1, LOXL2, and TGFB1 in relation to tumor infiltrating CTLs in non-small cell lung carcinoma (NSCLC). NSCLC tissue microarrays (TMAs) consisting of 63 independent patient FFPE tumor samples were analyzed using this ISH assay with the following probe combinations: Hs-CD8/Hs-IFNG, Hs-CD4/Hs-FOXP3, Hs-LOXL2/Hs-COL4A1, and Hs-TGFB1/Hs-COL11A1. We observed COL4A1 expression in both tumor and tumor-associated stromal cells in different samples. In contrast, COL11A1 was only expressed in tumor-associated stromal cells. Interestingly, high COL4A1 expression was associated with high CD8+ T cell infiltration, whereas high COL11A1 expression was associated with poor CD8+ T cell infiltration. In addition, tumor expression of TGFB1 was positively correlated with COL11A1 expression. These data depict a complex landscape of ECM gene expression and their relationship to T cell infiltration in the tumor and TME. Taken together, these results demonstrate that the RNAscope assay provides a powerful approach to directly examine the interactions between tumor, ECM, and T cell immune infiltration, and offers advantages over immunohistochemistry (IHC) for identifying the cellular sources of secreted proteins such as ECM components in the TME. Citation Format: NA LI, Hongzhe Sun, Xin Wang, Zhifu Zhang, Ying Zhou, Courtney Anderson, Xiao-Jun Ma. Extracellular matrix gene expression and cytotoxic T lymphocyte infiltration in the tumor microenvironment in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4965.

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines

Rabies is an infectious viral disease, characterized as a neglected zoonosis, responsible for nearly 60,000 deaths annually. The virus is transmitted mainly by dogs in Africa and Asia, and wildlife in Europe and the Americas, to all mammals’ species, causing severe encephalitis almost always fatal after the onset of neurological symptoms. Human rabies can be prevented through extensive vaccination of dogs and pre/post-prophylaxis treatments in humans with inactivated rabies vaccines. The vaccine manufacture involves a series of quality control assays using laboratory animals, which are mandatory to exclude the presence of viable residual virus. The quality controls must be carried out in various steps during the vaccine production, which demands the use of a large number of animals. In this study, we standardized a real-time quantitative RT-PCR duplex assay to be used during intermediate stages of the vaccine production. This assay was done for the quantification of vaccine strain rabies virus, targeting rabies nucleoprotein, and β-actin mRNA of BHK-21 cells as an internal endogenous control. The results showed specific amplification, with the analytical sensitivity ranged from 101 to 106 TCID50/mL with high repeatability rate for the quantification of rabies virus in inactivated vaccine samples. Global organizations are engaged to develop new approaches to determine viable residual virus, and this assay can be applied in combination with traditional in vitro methods for the release of intermediate batches of vaccines during the production process, keeping the in vivo tests only for final release.

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.

Mobile platform for rapid sub–picogram-per-milliliter, multiplexed, digital droplet detection of proteins

Digital droplet assays-in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually-have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are currently mainly confined to laboratory settings, due to the instrumentation necessary to serially generate, control, and measure tens of millions of droplets/compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology is based on three key innovations: (i) the integration and parallel operation of a hundred droplet generators onto a single chip that operates >100× faster than a single droplet generator, (ii) the fluorescence detection of droplets at >100× faster than conventional in-flow detection using time domain-encoded mobile phone imaging, and (iii) the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation. To demonstrate the power of this approach, we performed a duplex digital ELISA. We characterized the performance of this assay by first using spiked recombinant proteins in a complex media (FBS) and measured a limit of detection, 0.004 pg/mL (300 aM), a 1,000× improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system. We additionally measured endogenous GM-CSF and IL6 in human serum from n = 14 human subjects using our mobile duplex assay, and showed excellent agreement with the gold standard system ([Formula: see text]).

Establishment of a bead-based duplex assay for the simultaneous quantitative detection of Neuropilin-1 and Neuropilin-2 using xMAP technology and its clinical application.

BackgroundNeuropilins (Nrps) are a new type of broad‐spectrum tumor marker. Currently, a method for accurate simultaneous quantification of Nrps is not available. We aimed to develop a bead‐based and duplexed flow cytometric assay that could be used for accurate and simultaneous quantification of Nrp1 and Nrp2 for scientific research or clinical diagnosis.MethodsWe coupled anti‐human Nrp1‐11# mAb and anti‐human Nrp2‐C3 mAb to magnetic beads 18# and 25#, respectively. Capturing antibodies and detecting antibodies were then combined to detect Nrps by a bead‐based Luminex assay, which was subsequently applied to quantify Nrps in clinical serum samples.ResultsThe results showed that the detection value of Nrps ranged from 10 to 100 000 pg/mL for Nrp1 and from 25 to 100 000 pg/mL for Nrp2. The detection sensitivity reached 10 pg/mL for Nrp1 and 24.8 pg/mL for Nrp2. Intra‐assay variances ranged from 1.0% to 2.6% for Nrp1 and from 2.9% to 4.0% for Nrp2, and interassay variances ranged from 1.5% to 6.4% for Nrp1 and from 4.2% to 8.1% for Nrp2. The Nrp1 and Nrp2 recoveries were 96.6%‐103.6% and 95.6%‐102.3%, respectively. Irrelevant antigens had no interference in the paired‐detection system, and the mean fluorescence intensity (MFI) values were stable for months.ConclusionA bead‐based, duplexed flow cytometric assay (xMAP® technology) was developed to detect Nrp1 and Nrp2. The assay provided rapid, high‐throughput results and was much more sensitive, specific, reproducible, and stable than existing assays. In addition, this assay could be applied in early‐stage cancer screening, tumor malignancy analysis, and prognosis assessment.

Simultaneous detection of coconut lethal yellowing phytoplasmas (group 16SrIV) by real-time PCR assays using 16Sr- and GroEL-based TaqMan probes

Lethal yellowing (LY) represents a serious threat to coconut in Mexico, Central America and the Caribbean islands. It is caused by a phytoplasma of group 16SrIV, and particularly subgroups -A and -D have been the major phytoplasmas affecting coconut palms grown in the southeast of Mexico. Therefore, is important to reliably detect the 16SrIV group of phytoplasmas not only for diagnosis purposes but also to improve the understanding of pathogen-plant-vector pathosystems. The 16S ribosomal operon genes have been particularly used as the targets for probes and primers for universal phytoplasma detection. However, its conservative nature has imposed the search of non-ribosomal genes to support a finer characterization and differentiation of phytoplasmas, particularly those closely related and one alternative has been the GroEL gene for the specific detection of subgroups 16SrIV-A and 16SrIV-D. Sensitive, rapid and reliable singleplex and duplex assays were developed based on real-time PCR (TaqMan) targeting both the GroEL gene and 16S rRNA. The assay performed with higher sensitivity compared to conventional nested-PCR when analysing DNA extracts obtained from LY symptom bearing palms. The LY 16S TaqMan probe assays amplified DNA from both subgroups, while the LY GroEL TaqMan probe assay only amplified DNA from subgroup 16SrIV-A. The duplex TaqMan probe assay is a novel option for the simultaneous detection and quantification of LY phytoplasmas of 16Sr subgroups -A and -D, which are the phytoplasmas that predominantly affect palms in Mexico.

Development of a quantitative PCR method for screening ichthyoplankton samples for bigheaded carps

Monitoring ichthyoplankton is useful for identifying reproductive fronts and spawning locations of bigheaded carps (Hypophthalmichthys spp.). Unfortunately, sorting and identifying ichthyoplankton to monitor for bigheaded carp reproduction is time consuming and expensive. Traditional methods require frequent egg-larvae sampling, sorting of all samples to obtain presumptively identified bigheaded carp, and genetic validation of presumptively identified eggs. Quantitative PCR (qPCR) has the potential to streamline this process by identifying samples that likely do or do not contain a target species. Our objective was to develop a genetic screening tool using qPCR with the duplex assays SCTM4/5 and BHTM1/2 to prioritize samples that have a higher likelihood of containing bigheaded carp eggs or larvae. We used tandem ichthyoplankton samples collected for monitoring bigheaded carps in the Upper Mississippi, Illinois, and St. Croix rivers to evaluate the effectiveness of qPCR as a screening tool. Samples with > 10,000 copies of DNA had 100% occurrence of bigheaded carp eggs or larvae in the traditionally sorted samples, whereas samples with < 10 copies of DNA had 0% occurrence of ichthyoplankton from these invasive species. We used a logistic regression model to calculate the probability of finding bigheaded carp eggs or larvae based upon the number of DNA copies; 406 copies corresponded with a 50% probability of having bigheaded carp ichthyoplankton present in a sample. These data can be used to inform management actions (i.e., control, containment) for these invasive fishes, and this tool could be adapted for monitoring for reproduction of other aquatic invasive species.

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.

Ultra-Fast On-Site Molecular Detection of Foodborne Pathogens Using a Combination of Convection Polymerase Chain Reaction and Nucleic Acid Lateral Flow Immunoassay.

Rapid detection and timely treatment of diseases caused by foodborne pathogens is important for improving the curative efficiency and preventing the spread of disease. In this study, we developed an assay utilizing a recently introduced ultra-fast convection polymerase chain reaction (cPCR) in conjunction with a simple nucleic acid lateral flow (NALF) immunoassay for ultra-fast on-site molecular detection of foodborne pathogens. Two Salmonella enterica serovars, Salmonella Enteritidis and Salmonella Typhimurium, and Escherichia coli O157:H7 were used as the target pathogens. We confirmed the specific amplification of the target species with specifically designed modified primer sets for cPCR in singleplex and duplex modes. After cPCR amplification, we compared the detection specificity and sensitivity using agarose gel electrophoresis and NALF assays with one or two test lines. The cPCR amplicons were readily and sensitively detected using the NALF assay, and the sensitivity was comparable with that of agarose gel electrophoresis. To confirm the application of the assay in real-life samples, the assay was used to test artificially contaminated milk. Without sample pre-enrichment, the limit of detection (LOD) was 4.5 × 103 colony-forming units (CFU)/mL for Salmonella species; 4.5 × 104 CFU/mL to differentiate Salmonella Enteritidis and Salmonella Typhimurium; and 2.3 × 103 CFU/mL for E. coli O157:H7, in a duplex assay. With a 6 h pre-enrichment, the LOD was 4.5 CFU/mL for Salmonella Enteritidis and Salmonella Typhimurium, and 2.3 CFU/mL for E. coli O157:H7. The cPCR amplification took only 14 min, and the NALF assay took ca. 5 min. The total analysis time was less than 20 min. Based on these observations, we propose that the developed assay is simple, ultra-fast, and applicable for on-site detection of foodborne pathogens.

Development of a triplex mtDNA qPCR assay to assess quantification, degradation, inhibition, and amplification target copy numbers

A hybrid absolute/relative qPCR assay which provides information regarding the condition of mitochondrial DNA (mtDNA) in a DNA sample is described. MtDNA concentration (copy number/μL) is determined via absolute quantification using a standard curve of a synthetic duplex DNA previously described (Kavlick et al., 2011). The state of mtDNA degradation is determined via the relative quantification of a mtDNA target found within the 16 s rRNA gene which is 3× longer than that of the short target in the former duplex assay, using the delta, delta Ct (ΔΔCt) method. The presence or absence of PCR inhibitors in the sample is qualitatively determined using a custom internal positive control (IPC) system which targets a unique and non-naturally occurring duplex DNA sequence. This IPC effectively detected inhibition by humic acid, tannic acid, melanin, and EDTA. All three assay components utilize sensitive and specific hydrolysis probes. The utility of ΔΔCt method was demonstrated in a series of experiments involving laboratory-fragmented DNA. Also described is a method for estimating copy number of any mtDNA target longer than the two targets amplified. The described triplex assay works well for intact and for fragmented or degraded mtDNA and therefore may be useful in forensic and ancient DNA disciplines as well as in biomedical research or practice.

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food

Meat products are prone to adulteration by the replacement of meat from more expensive animal species with meat from cheaper sources. We present a DNA metabarcoding method allowing the identification and differentiation of 15 mammalian and six poultry species in foodstuffs. The method, developed on the MiSeq® platform, targets a mitochondrial 16S rDNA region recently found to be suitable for the differentiation of 300 mammalian species. We designed a novel primer pair for poultry and applied it in combination with the primer pair for mammalian species in a duplex assay. The applicability of the method was investigated by analysing DNA extracts from muscle, DNA extract mixtures and extracts from model sausages. Our results indicated that the species of interest can be identified, differentiated and detected down to a proportion of 0.1%. Since 96 samples can be sequenced in one run, the method has high potential for application in routine analysis.

Rapid diagnostic tests relying on antigen detection from stool as an efficient point of care testing strategy for giardiasis and cryptosporidiosis? Evaluation of a new immunochromatographic duplex assay

Microscopy is the gold standard for the diagnosis of gastrointestinal parasites but is time-consuming and dependent on operator skills. Rapid diagnostic tests represent alternative methods but most evaluations have been conducted on a limited number of samples preventing their implementation in the clinical setting. We evaluated a new CE-IVD marked immunochromatographic assay (Crypto/Giardia K-SeT®, Coris Bioconcept) for the detection of G. intestinalis and Cryptosporidium spp. in 2 phases (retrospective and prospective) on a set of 482 stool samples including rare Cryptosporidium species. Besides G. intestinalis, this test could represent a rapid and reliable alternative to the modified Ziehl-Neelsen staining for the diagnosis of cryptosporidiosis (sensitivity/specificity were 89.2%/99.3% and 86.7%/100% for G. intestinalis and Cryptosporidium resp.), reducing diagnostic delays. Such strategy would also be time-saving by avoiding wet mount microscopy and concentrations steps, being particularly appropriate for laboratories having little expertise in microscopy or not able to implement molecular diagnostic methods.

Abstract 5531: Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAF mutant metastatic melanoma undergoing treatment with BRAF-targeted therapies

Abstract Purpose Melanoma patients with BRAFmutant tumors often develop resistance to BRAF-inhibitor therapies. Co-occurring mutations, present at the time of treatment initiation, have been identified in patients with primary treatment resistance, and NRAS mutations have been associated with secondary resistance. We tested the ability of multiplex mutation detection assays to identify possible co-occurring and resistance mutations in cell-free, circulating tumor DNA (ctDNA) from patients undergoing treatment with BRAF-targeted therapies. Methods Purified ctDNA samples remaining from a previous longitudinal study of metastatic melanoma patients, which measured BRAFmutant and NRASmutant ctDNA using droplet digital PCR (ddPCR) duplex assays, were used for this study. Twelve samples from 6 patients with BRAF V600E mutant (V600E) tumors, who were receiving BRAF-inhibitor therapy, were separately analyzed in different laboratories using the two different multiplex assays (Oncomine and OnTarget). Oncomine used approximately 20ng DNA; OnTarget used approximately 30ng DNA. Patient samples were chosen based on clinical response or disease progression at the time of blood draw. Results Ten of 12 samples had V600E ctDNA detected by ddPCR. The On-Target detected V600E in 9/10 samples; the Oncomine detected V600E in 8/9 samples (1 sample was not analyzed due to limited sample availability). The 1 sample with a ddPCR detected mutation that was not detected by either OnTarget or Oncomine had a 0.01% fractional abundance, which is below the detection limit of these multiplex assays. Neither assay detected V600E DNA when it was not detected by ddPCR. One patient had 2 samples with a co-occurring p53 (R273H) mutation detected by OnTarget and Oncomine. This mutation was present at the time of partial response, and at disease progression with an associated increased fractional abundance as determined by both assays. The OnTarget detected NRASmtant ctDNA (Q61K x 2, Q61H x1) in 3 of the V600E samples; the Oncomine detected the Q61K in 1 of these 2 samples (the third sample was not analyzed as noted above). The NRAS mutations arose at times of disease progression. Oncomine also detected additional p53, GNAS, and FBXW7 mutations at generally low fractional abundances that were not detected by OnTarget. Conclusion Co-occurring and potential resistance mutations are detectable in the plasma of metastatic melanoma patients using OnTarget or Oncomine assays. The assays demonstrate high sensitivity, as evidenced by their ability to identify V600E ctDNA. These findings suggest that studies analyzing patient ctDNA for resistance mutations while they are undergoing treatment are feasible. Results of such studies may eventually help inform treatment choices, such as switching therapies when resistance mutations emerge. Citation Format: Broderick Corless, Greg Chang, Weihua Liu, Jin Li, Andre Marziali, Laura Mai, Matthew Wiggin, Melissa Wilson, Anna Pavlick, Iman Osman, George Karlin-Neumann, Cindy Spittle, David Polsky. Detection of co-occurring and potential resistance mutations in cell-free, circulating tumor DNA from patients with BRAFmutant metastatic melanoma undergoing treatment with BRAF-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5531.

An automated approach to classification of duplex assay for digital droplet PCR.

In the digital polymerase chain reaction (dPCR) detection process, discriminating positive droplets from negative ones directly affects the final concentration and is one of the most important factors affecting accuracy. Current automated classification methods usually discuss single-channel detections, whereas duplex detection experiments are less discussed. In this paper, we designed a classification method by estimating the upper limit of the negative droplets. The right tail of the negative droplets is approximated using a generalized Pareto distribution. Furthermore, our method takes fluorescence compensation in duplex assays into account. We also demonstrate the method on Bio-Rad's mutant detection dataset. Experimental results show that the method provides similar or better accuracy than other algorithms reported over a wider dynamic range.