Triplex 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.

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

Food and medicinal plants from Nigeria with anti-Helicobacter pylori activities induce apoptosis in colon and gastric cancer cell lines

Background: Food and medicinal plants are used traditionally in Nigeria to treat gastrointestinal (GI) disorders such as gastritis, peptic ulcer disease and GI-related cancers. We have previously reported that specific extracts of Nigerian food and medicinal plants inhibited the growth of Helicobacter pylori, a bacterium known to cause peptic ulcer disease, as well as gastric and colon cancer. Objective: To determine the effects of three food plants, namely Anogeissus leiocarpus (DC.) Guill. &amp; Perr. (African birch, Combretaceae), Terminalia glaucescens Planch ex Benth. (Nigerian chewing sticks, Combretaceae) and Dillenia indica L. (Elephant apple, Dilleniaceae) used in Nigeria for the treatment of GI disorders and cancer in six colon and gastric cancer cell lines, and two non-cancerous cell lines. Methods: Cancer cells were grown in appropriate media and CellTiter-Glo® 2.0 and ApoTox-Glo™ Triplex assays were used to measure cell growth and apoptosis in SW480, SW620, HCT116, Caco2 cell lines, as well as AGS and NCI-N87 gastric cancer cells. Caspase-Glo® 3/7, and Caspase-Glo® 8 were used to determine caspase activities and apoptosis. Gene expression was measured using quantitative PCR.Results: The methanol extract of A. leiocarpus roots inhibited the growth of HCT-116, SW480 and SW620 colon cancer cells (IC50 of 15.8, 10.0, and 20.8 µg/mL, respectively), but weakly active in the AGS and NCI-N87 cells (IC50 77.0 and &gt;100 µg/mL respectively). The T. glaucescens extract was weakly active in HCT-116 cells (IC50 64.9 µg/ml) and AGS cells (52.2 µg/ml). The D. indica extract was active in SW480 (IC50 15.8 µg/mL), and weakly active in Caco2 (IC50 35.2 µg/mL) and HCT-116 (IC50 53.2 µg/mL). In HCT-116 cells, A. leiocarpus root extract and its aqueous partition increased caspase 8 activity, as well as mRNA expression of p53, while expression of Bcl-2 and HDAC1 mRNA was reduced. Bioassay guided fractionation of the aqueous partition led to the isolation and identification of the known compound methyl gallate.Conclusions: These data suggest that the Nigerian food plants with anti-Helicobacter pylori activities increase the expression of the tumor suppressor p53 and inhibit HDAC1, thereby inducing apoptosis in HCT-116 colon cancer cells.Running Title: Inhibitory Effects of Nigerian food plants on GIT CancersKeywords: African birch, AGS, Apoptosis, Anogeissus leiocarpus, Dillenia indica, Elephant apple, Terminalia glaucescens, Nigerian chewing sticks, HCT-116, SW480, SW620

Quadruplex Real-Time TaqMan® RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes.

Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission.

Effect of NLRP3 inflammasome genes polymorphism on disease susceptibility and response to TNF-α inhibitors in Iraqi patients with rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a genetically predisposed, systemic, chronic, inflammatory disease. Immune system dysregulation and inherited susceptibility polymorphisms suggest that this type of variation is functional and may help predict disease susceptibility and develop new therapeutic strategies. Anti-TNF-alpha (TNF-α) drugs are highly effective RA treatments, but not all patients respond the same way. It's important to figure out whether RA risk alleles can identify and predict anti-TNF-α-responsiveness in RA patients. Aims of the studyExamine the function of the NLR family pyrin domain containing 3 (NLRP3) and caspase recruitment domain family member 8 (CARD8) genes polymorphisms and their morbid genotypes and alleles in RA patients and apparently healthy controls. In addition, their role in disease susceptibility, severity, and response to anti-TNF-α therapy. Also, examine how single nucleotide polymorphisms (SNPs) affect serum levels of pro-inflammatory cytokines like TNF-α and interleukin (IL)-1β. Materials and methods100 RA patients (88 females, 12 males) and 100 apparently healthy people (86 females, 14 males) were examined. To measure serum TNF-α and IL-1β, Elabscience sandwich ELISA kits were used. Iraq Biotech, Turkey DNA extraction kit was used to extract genomic DNA from whole blood. CARD8 (rs2043211) and NLRP3 (rs4612666) were genotyped using Agilent, AriaMx, USA, through Tri-Plex SYBR Green-based real-time PCR allelic discrimination assays. Geneious software, version 2019.2.2, used to design primers from published sequences (GenBank accession no. GCA 009914755.1). Primer specificity was determined by NCBI's BLAST. ResultsStudy found that there is association between cytokines serum level and 28-joints disease activity score (DAS-28). The level of TNF-α increases with the higher DAS-28 (r2 = 0.45, P < 0.0001). Also, IL- 1β level increases with higher DAS-28 (r2 = 0.51, P < 0.0001). There were no statistically significant variations between patients with RA and the control group in the distribution of CARD8 SNP rs2043211 and NLRP3 SNP rs4612666 genotypes (P = 0.17 and 0.08 respectively) as well their alleles (P = 0.059 and 0.879 respectively). CARD8 (rs2043211) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL-1β serum levels (P < 0.0001 for both). Also, NLRP3 (rs4612666) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL- 1β serum levels (P < 0.0001 for both). Interestingly, this study revealed that CARD8 (rs2043211) and NLRP3 (rs4612666) variant genotypes are associated with lower response to anti-TNF-α drugs. ConclusionsSerum TNF-α and IL-1β correlate with DAS-28 and disease activity. Non-responders have elevated TNF-α and IL-1β. CARD8 rs2043211 and NLRP3 rs4612666 variant polymorphisms are associated with high serum TNF-α and IL-1β, active disease course, poor disease outcomes, and low response to anti-TNF-α therapy.

Combinations of vitamin A and D induced are synergistic in breast cancer cells and alter gene expression in the endoplasmic reticulum stress, unfolded protein and estrogen signaling canonical pathways

Introduction: Numerous studies over the past 30 years have shown that bioactive compounds present in functional foods, including vitamins, minerals and phytochemicals reduce cancer risk. For example, vitamins A and D derivatives found in fruits, vegetables, and dairy products, reduce the growth of breast, bladder, head, neck, lung, prostate, and skin cancers. However, the effects of these combined vitamins have not been previously reported for breast cancer.Aims: To investigate the activities of vitamin A (all-trans-retinoic acid; ATRA), as well as vitamins D2 and D3 in combination in the breast epithelial cancer cell lines T47D:A18, MCF-7, and SK-BR-3 and perform whole genome analysis in MCF-7 cells using RNA-seq.Methods: Breast cancer cells were cultured in appropriate media and treated with ATRA, D2 or D3 alone in concentrations from 1-10 μg/ml, or in combination at 1, 5, and 10 μg/ml. The CellTiter-Glo® 2.0 assay, Caspase-Glo®3/7, Caspase®8, and ApoTox-Glo™ Triplex assays measured cell viability and apoptosis. The effect of treatment on autophagy in MCF-7 cells was measured with a CYTO-ID® Autophagy Detection Kit 2.0. The whole transcriptome analysis was assessed using mRNA-seq and qPCR.Results: Separately, ATRA, D2, and D3 all reduced the viability of all breast cancer cell lines tested, with median inhibitory concentrations (IC50) between 2.1 to 31.7 μg/ml. However, when breast cancer cells were treated with combinations of ATRA+D2+D3, the IC50 was reduced indicating synergism. In MCF-7 cells, 5-flurouracil (5-FLU) had an IC50 of 1.37 µg/ml, while the vitamin A and D combination had an IC50 of 1.5 µg/ml, indicating the combination was ~90% as effective as 5-FLU. Treatment of MCF-7 cells with ATRA+D2+D3 enhanced caspase 3/7 activity, as well as the expression of Bax, BAD, PTEN and p53 (apoptosis canonical pathway), as well as induced autophagy. Whole genome analysis of treated MCF-7 cells showed a significant upregulation in gene expression in the autophagy, endoplasmic reticulum stress and the unfolded protein response apoptosis canonical pathways. Furthermore, MCF-7 cells treated with ATRA+D2+D3 demonstrated significant downregulation of gene expression in estrogen-mediated S phase entry and estrogen signaling canonical pathways suggesting antiestrogenic effects. Conclusions: Vitamins A and D combinations had synergistic effects in breast cancer cells and induced both apoptosis and autophagy. Transcriptional profiling showed significant alterations in gene expression patterns and upregulation of multiple cancer signaling pathways supporting the hypothesis that combining vitamins A and D is a more effective treatment than either vitamin alone. Keywords: all-trans-retinoic acid (ATRA), Vitamin D2, Vitamin D3, breast cancer cells, apoptosis, estrogen receptors

Table S7. Nucleotide sequences used in triplex assays

Table S7. Nucleotide sequences used in triplex assays

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.

Multiplex Mass Spectrometry for Enzymatic Activities Screening of Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) are a group of rare and inherited disorders of lysosome function and they are classified into more than 50 types (e.g., mucopolysaccharidoses, neuronal ceroid lipofuscinoses, Fabry’s Disease; Pompe Disease, etc.). The majority of LSDs result from defective lysosomal enzyme activity leading to accumulation of endogenous macromolecules in the lysosome causing severe disease symptoms and finally death. For some LSDs, treatment is available in the form of enzyme replacement therapy with good results if the therapy is started as soon as possible, making the diagnosis of crucial importance. State of art now in LSDs diagnostic employs in first stage, enzymatic determinations by fluorimetry or by mass spectrometry (in few cases).In this report, we describe a highly specific and sensitive diagnostics on dry blood spots (DBSs) for (i) molecular determinations of LSDs, particularly mucopolysaccharidoses (MPSs) and neuronal ceroid lipofuscinoses (NCLs), by simultaneous fluorimetric and mass spectrometric analysis using new types of substrate derivatives based on different alkyl‐umbelliferone; (ii) clinical diagnostics of LSDs by multiplex‐MS‐MRM analysis using new type of substrate derivatives and (iii) clinical validation of the new developed methods using DBS samples for patients with established LSDs. We have developed a high‐throughput single and multiplexing assay for rare diseases (e.g. MPS 1, MPS 2, MPS 6, NCL 2, NCL 10, Fabry Disease, etc.) in DBSs. A common substrate for the fluorimetric measurement of a lysosomal enzyme activity is a molecule with 2 different parts which are bound covalently: a group recognized by the target enzyme (e.g. sugar, lipid) and 4‐umbelliferone and the product resulted after the enzymatic reaction is always 4‐methyl umbelliferone. In our tests, the enzymatic activity levels in DBSs were determinate by fluorimetry or multiple reactions monitoring mass spectrometry in the presence of an internal standard showing a good statistical correlation in singles assays. Moreover, we developed duplex and triplex assays for the diagnosis of different LSDs from the muccopolysacharidoses family using modified substrates based on different coumarin derivates obtained through Pechman condensation. The new umbelliferone derivatives were synthesize by substituting groups in position 4 and 7 of the alpha‐ coumarin molecule with others e.g. 4‐ ethyl‐ umbelliferone, 4‐propyl‐umbelliferone, etc.In conclusion, by quantification of umbelliferone derivatives as products of enzymatic reactions either by fluorimetry or mass spectrometry, the newly proposed substrates can be easily use for clinical diagnosis of several LSDs.

Direct Real-Time PCR for the Detection and Serotyping of Haemophilus influenzae without DNA Extraction.

To monitor the burden and changes in Haemophilus influenzae (Hi) disease, direct real-time PCR (drt-PCR) assays have been developed for Hi detection in monoplex form and its six serotypes in triplex form, directly from cerebrospinal fluid (CSF) specimens. These assays target the phoB gene for the species detection (Hi-phoB) and serotype-specific genes in region II of the capsule biosynthesis locus (Hi-abf and Hi-cde), identified through comparative analysis of Hi and non-Hi whole-genome sequences. The lower limit of detection (LLD) is 293 CFU/mL for the Hi-phoB assay and ranged from 11 to 130 CFU/mL for the triplex serotyping assays. Using culture as a reference method, the sensitivity and specificity of Hi-phoB, Hi-abf, and Hi-cde were 100%. Triplex serotyping assays also showed 100% agreement for each serotype compared to their corresponding monoplex serotyping assay. These highly sensitive and specific drt-PCR assays do not require DNA extraction and thereby reduce the time, cost, and handling required to process CSF specimens. Furthermore, triplex drt-PCR assays combine the detection of three serotypes in a single reaction, further improving testing efficiency, which is critical for laboratories that process high volumes of Hi specimens for surveillance and diagnostic purposes.

Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR.

Simple SummaryDigital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantification of two, three, and four GM canola and soybean events at the same time using the QX200 Droplet Digital PCR (ddPCR) system. Adjustments of the probe concentrations and labels for some of the assays were needed for successful multiplex ddPCR. Absolute quantification of GM canola and soybean events was achieved for duplex, triplex, and tetraplex ddPCR at 0.1%, 1%, and 5% concentrations.The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection.

Extracts of Anogeissus leiocarpus and Dillenia indica Inhibit the Growth of MCF-7 Breast Cancer and COV434 Granulosa Tumor Cells by Inducing Apoptosis and Autophagy

Background: Dillenia indica L. (Dilleniaceae) and Anogeissus leiocarpus (DC.) Guill. &amp; Perr. (Combretaceae) are used in traditional Nigerian medicine to treat various forms of cancer. This study investigated the cytotoxic effects of these plant extracts using COV434 granulosa tumor and MCF-7 breast cancer cells. Methods: Samples of D. indica and A. leiocarpus were collected in Ibadan, Nigeria, air-dried, and extracted with methanol. Cell viability and cytotoxicity were determined using CellTiter-Glo® 2.0 assay at concentrations from 1 to 100 μg/mL. Caspase activity and apoptosis were determined using Caspase-Glo® 3/7, Caspase-Glo® 8, and ApoTox-Glo™ triplex assays, and qPCR. Autophagy was measured using a Cyto-ID Autophagy Detection Kit. Results: In COV434, aqueous partitions of A. leiocarpus root (ALR-Aq) and stem bark (ALS-Aq) had IC50s of 23.5 and 26.7 μg/mL, respectively. In MCF-7 cells, the ALR MeOH extract had IC50 of 12.75 μg/mL, while the DIS-Aq had IC50 of 65.28 μg/mL. None of the extracts inhibited the growth of human osteoblasts or rat myoblasts at similar concentrations. Treatment with ALR-Aq and DIS-Aq induced mitochondrial apoptosis in MCF-7 and COV434. Both ALR-Aq and DIS-Aq induced autophagy in COV434 cells, while ALR-Aq induced autophagy in MCF-7 cells. Ellagic acid (IC50 of 3.27μg/mL in COV434 cells) was isolated from ALR-Aq using bioassay-guided fractionation. Conclusion: DIS-Aq and ALR-Aq induced apoptosis in MCF-7 and COV434 cancer cells. Ellagic acid was isolated as the active constituent. Taken together, these data suggest that both plant extracts have strong anti-proliferative effects, and further investigation for their anticancer effects is warranted.

Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2, and RP targets for SARS-CoV-2 diagnosis

BackgroundSeveral RT-qPCR kits are available for SARS-CoV-2 diagnosis and some have emergency use authorization from the US Food and Drug Administration. In particular, the nCoV19 CDC kit includes two targets for detecting SARS-CoV-2 (N1 and N2) and an RNaseP (RP) target for RNA extraction quality control, all of which are labeled with FAM, and thus three PCR reactions are required per sample. MethodsWe designed a triplex RT-qPCR assay based on nCoV19 primers and probes where N1, N2, and RP are labeled with FAM, HEX, and Cy5, respectively, so only a single PCR reaction is required for each sample for SARS-CoV-2 diagnosis. ResultsIn total, 172 samples were analyzed in both singleplex and triplex assays, where 86 samples tested SARS-CoV-2 negative with both assays, so the triplex assay specificity was 100%. In addition, 86 samples tested SARS-Co-V 2 positive with the singleplex assay and 84 with the triplex assay, so the sensitivity was 97.7%. The limit of detection for the triplex assay was determined as 1000 copies/mL. ConclusionsThis new triplex RT-qPCR assay based on primers and probes from the CDC protocol is highly reliable for SARS-CoV-2 diagnosis, and it could speed up detection and save reagents during the current SARS-CoV-2 testing supplies shortage.

Triplex Real-Time PCR without DNA Extraction for the Monitoring of Meningococcal Disease.

Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178–5264 CFU/mL by monoplex and 68–2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.

Evaluation of Two Triplex One-Step qRT-PCR Assays for the Quantification of Human Enteric Viruses in Environmental Samples

Human enteric viruses are responsible for waterborne and shellfish-associated disease outbreaks worldwide. Quantitative reverse transcription PCR (qRT-PCR) is often used to assess the health risks associated with shellfish and environmental water, but viral titres in sediments are less commonly investigated. In this study, we developed and validated two multiplex qRT-PCR assays for aquatic sediment and shellfish samples targeting viruses that are a common cause of gastroenteritis (norovirus GI, GII and hepatitis A virus), two emerging viruses (sapovirus and hepatitis E virus), along with mengovirus (MgV), which is often used as a sample process control for the assessment of RNA extraction efficiency. Singleplex and multiplex assays demonstrated comparable PCR efficiencies and gave reliable results over a wide concentration range. The multiplex assays showed remarkable sensitivity with a limit of detection of 1 RNA copy/µL nucleic acid extract for all target viruses and limits of quantification of 3–18 RNA copies/µL for the targeted human pathogenic viruses and 20–40 RNA copies/µL for MgV. The results demonstrated the veracity of multiplex qRT-PCR for the estimation of viral titres in sediment and shellfish, allowing the rapid assessment of viral infection risks associated with environments exposed to wastewater contamination.

Triplex tandem mass spectrometry assays for the screening of 3 lysosomal storage disorders in a Korean population

BackgroundWe evaluated the performance of triplex tandem mass spectrometry (MS/MS) assays using dried blood spots (DBSs) to screen for 3 lysosomal storage disorders (LSDs): Pompe, Fabry, and Gaucher diseases. MethodsAn Acquity UPLC CSH C18 column (Waters) and a TQD triple quadrupole mass spectrometer (Waters) in the multiple reaction monitoring (MRM) mode were used to evaluate the precision, carryover, and ion suppression of 3 enzyme assays (acid alpha glucosidase, acid α-galactosidase, and acid β-glucocerebrosidase) and were used to screen 1606 anonymous newborn DBSs. ResultsIntra- and inter-run CVs were between 0 and 14.1% and, between 0 and 18.9%. The carryover was 0%, −0.14%, and 0.39% for the 3 enzyme activities, respectively. No ion suppression was observed. The data from the 1606 anonymous newborn DBSs exhibited approximately bell-shaped distributions of 3 enzymatic activities (the median values were 16.46μmol/h/l, 7.25μmol/h/l, and 25.63μmol/h/l, respectively). ConclusionsThe performance of the triplex tandem mass spectrometry assays for the screening of 3 lysosomal storage disorders in a Korean population using DBSs was acceptable.

Abstract 5058: Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors

Abstract Introduction: Androgen receptor (AR) signaling plays a critical role in all the stages of prostate cancer (CaP) ranging from organ confined to castration-resistant (CRPC) phases. Although androgen deprivation therapy (ADT) remains the mainstay treatment for advanced CaP, the inevitable transition from androgen- sensitive to CRCP presents the most significant challenge in CaP therapy. Androgen dependent expression of oncogenic ETS related gene (ERG) in half of all CaP in western countries plays critical role in the tumorigenesis of CaP through regulation of cancer specific signaling pathways. We found that NOTCH transcription factors are common targets of ERG in ERG positive cancer cells. NOTCH signaling pathway is an important signaling pathway in the development of drug-resistant tumor growth. In the current study we evaluated the combinatorial effects of NOTCH and AR inhibitors in the context of ERG positive prostate cancer cells. Methods: ERG, NOTCH1, NOTCH2 and downstream targets of NOTCH transcription factors were analyzed by Western blot assays. Dose and time kinetics of combining NOTCH inhibitor (γ-Secretase inhibitor 1, GSI-1) and AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) were assessed in a panel of ERG positive or ERG negative CaP cells. Trypan blue exclusion, methylthiazole tetrazolium (MTT), or ApoTox-Glo™ Triplex assays were used to asses cell proliferation, apoptosis and drug cytotoxicity. Results: Prostate cancer cell lines with endogenous or ectopic expression of ERG showed upregulation of NOTCH1 and NOTCH2. The NOTCH inhibitor, GSI-1 conferred an increased sensitivity to all tested AR inhibitors (Bicalutamide, Enzalutamide, and Abiraterone) with bicalutamide showing the most robust inhibition of AR, ERG, NOTCH1, NOTCH2, PSA, decreased cell growth and enhanced apoptosis in ERG positive VCaP cells. This observation was not seen in ERG negative LNCaP cells or in ERG positive primary endothelial cells. Conclusions: NOTCH inhibitor enhanced sensitivity of AR inhibitors in ERG positive VCaP cells growth. The combination of the GSI-1 with AR inhibitors has shown synergistic effect when compared to single agent treatment. Taken together, our study suggests that NOTCH inhibitors may enhance the actions of AR inhibitors in the treatment of ERG positive prostate cancers. Inhibition of AR and NOTCH signaling may offer new opportunities in assessing ERG targeted therapy for prostate cancer. Citation Format: Ahmed A. Mohamed, Shyh-Han Tan, Shilpa Katta, Charles P. Xavier, Lakshmi Ravindranath, Wei Huang, Hua Li, Meera Srivastava, Shashwat Sharad, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, Shiv Srivastava. Silencing of NOTCH signaling enhances the sensitivity of ERG positive prostate cancer cells to AR inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2015-5058

Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR

Background: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs.Objectives: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method.Results: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates.Conclusions: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.

Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases.