Signal Amplification Assay Research Articles

DNA-Functionalized Gold Nanorods for One-Step Simultaneous Determination of Multiple microRNAs Based upon Plasma-Enhanced Fluorescence

MicroRNAs (miRNAs) are endogenous, noncoding, and sequence-specific single-stranded RNA regulatory factors whose abnormal expression levels are closely related to various diseases, especially cancer, and are considered effective biomarkers for disease diagnosis and prognosis. Since the concentrations of miRNAs in the body are low and disease diagnosis by detecting single miRNAs may result in a high false positive, it is essential to construct a sensitive biosensor for the determination of multiple miRNAs. In this work, an enzyme-free signal amplification assay based on plasma-enhanced fluorescence (PEF) is reported for the one-step simultaneous determination of two miRNAs. Gold nanorods (AuNRs) were functionalized with fluorescein isothiocyanate (FAM)-tagged hairpin DNA1 (H1) and cyanine 5.5 (Cy5.5)-tagged hairpin DNA2 (H2). The fluorescence signals of FAM and Cy5.5 were quenched due to their proximity to the surface of the AuNRs. In the presence of target miRNA-21 and miRNA-155, H1 and H2 complemented miRNA-21 and miRNA-155, respectively, and the fluorescence signals of FAM and Cy5.5 were not only restored but amplified due to the plasma-enhanced fluorescence. A suitable linear range (0.1 to 10 nM) and low limits of detection for miRNA-21 (23 pM) and miRNA-155 (1.6 pM) were achieved. The developed assay for the one-step simultaneous measurement of two miRNAs is simple, fast and sensitive, exhibiting great potential for monitoring these biomarkers.

Efficient magnetic enrichment cascade single-step RPA-CRISPR/Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100-fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPA-CRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/μL (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signal-amplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.

Construction of an Endogenously Activated Signal Amplification Assay for In Situ Imaging of MicroRNA and Guided Precise Photodynamic Therapy for Cancer Cells.

The construction of a sensitive strategy for in situ visualizing and dynamic tracing intracellular microRNA is of great importance. Via the toehold-mediated strand displacement process, the catalytic hairpin assembly (CHA) could offer several hundreds-fold signal amplifications. However, the CHA may produce certain background interferences since microRNA may exist in normal cells. In this work, we constructed an endogenously and sequentially activated signal amplification strategy to provide the amplified dual-color fluorescence imaging of microRNA in living cancer cells, which was comprised of two successive reaction processes: the activation of the preprotective catalytic probe by the endogenous glutathione (GSH) and the subsequent catalytic hairpin assembly on the surface of the upconversion nanoprobe triggered by the specific microRNA. Since the concentration of GSH in cancer cells was much higher than that in normal cells and the extracellular environment, the activation of the designed nanoprobe could be controlled at the desirable site. With the merits of the endogenous initiation and selective activation, the designed nanoprobe could achieve the bioimaging of microRNA in living cancer cells with high precision and reliability. Furthermore, via the introduction of a photosensitizer molecule into the DNA strand, the designed nanoplatform could achieve the precise photodynamic therapy (PDT) for cancer cells and malignant tumors under the irradiation of the NIR laser. This work provided a new avenue to achieve the accurate imaging of intracellular microRNA and guided precise PDT, which would offer powerful hints to the early diagnosis and therapy of malignant tumors.

A low-cost, paper-based hybrid capture assay to detect high-risk HPV DNA for cervical cancer screening in low-resource settings.

Cervical cancer is a leading cause of cancer death for women in low-resource settings. The World Health Organization recommends that cervical cancer screening programs incorporate HPV DNA testing, but available tests are expensive, require laboratory infrastructure, and cannot be performed at the point-of-care. We developed a two-dimensional paper network (2DPN), hybrid-capture, signal amplification assay and a point-of-care sample preparation protocol to detect high-risk HPV DNA from exfoliated cervical cells within an hour. The test does not require expensive equipment and has an estimated cost of <$3 per test without the need for batching. We evaluated performance of the paper HPV DNA assay with short synthetic and genomic HPV DNA targets, HPV positive and negative cellular samples, and two sets of clinical samples. The first set of clinical samples consisted of 16 biobanked, provider-collected cervical samples from a study in El Salvador previously tested with careHPV and subsequently tested in a controlled laboratory environment. The paper HPV DNA test correctly identified eight of eight HPV-negative clinical samples and seven of eight HPV-positive clinical samples. We then performed a field evaluation of the paper HPV DNA test in a hospital laboratory in Mozambique. Cellular controls generated expected results throughout field testing with fully lyophilized sample preparation and 2DPN reagents. When evaluated with 16 residual self-collected cervicovaginal samples previously tested by the GeneXpert HPV assay ("Xpert"), the accuracy of the HPV DNA paper test in the field was reduced compared to testing in the controlled laboratory environment, with positive results obtained for all eight HPV-positive samples as well as seven of eight HPV-negative samples. Further evaluation showed reduction in performance was likely due in part to increased concentration of exfoliated cells in the self-collected clinical samples from Mozambique compared with provider-collected samples from El Salvador. Finally, a formal usability assessment was conducted with users in El Salvador and Mozambique; the assay was rated as acceptable to perform after minimal training. With additional optimization for higher cell concentrations and inclusion of an internal cellular control, the paper HPV DNA assay offers promise as a low-cost, point-of-care cervical cancer screening test in low-resource settings.

Label-free and highly sensitive detection of CRP based on the combination of nicking endonuclease-assisted signal amplification and capillary electrophoresis-UV assay

Elevated C-reactive protein (CRP) levels are linked with bacterial infection, local inflammation in osteoarthritis and the increased risk of developing cardiovascular disease. Here, a sensitive and label-free CRP assay is developed by combining cyclic enzymatic signal amplification and capillary electrophoresis (CE) with UV detection. This assay is constructed of base pairing and target recognition. Thereinto, nicking endonuclease (NEase) can recognize the specific nucleotide sequences in double-stranded DNA (dsDNA), which is formed by a CRP aptamer and its complementary DNA (cDNA). Sequentially, NEase cleaves only cDNA to produce signal DNAs. Therefore, a large number of signal DNAs are generated through continuous enzyme cleavage. In the presence of CRP, the aptamer recognizes and binds to CRP with high affinity and selectivity, which results in a decrease in signal DNAs, and thus the UV absorption value of CE significantly decreases, too. A wide linear range was obtained between 0.0125 and 15 μg mL−1 (0.11–130.5 nM) in 1% human serum with a detection limit of 4 ng mL−1 (35 pM). Additionally, the proposed method is universal and can be applied to analyze other similar substances by altering the matched aptamer.

A Target-Feedback Rolling-Cleavage signal amplifier for ultrasensitive electrochemical detection of miRNA with Self-Assembled CeO2@Ag hybrid nanoflowers

Currently, developing an effective and easy-to-operate signal amplification assay to detect the trace-amount miRNAs in serum remains a significant challenge. Herein, an ultrasensitive CeO2@Ag hybrid nanoflower (CeO2@Ag HNF)-labeled electrochemical biosensor was developed for sensing miRNA, based on a target-feedback rolling-cleavage (TFRC) signal amplifier. CeO2@Ag HNFs possessing a unique three-dimensional layered structure were synthesized without any complex reaction conditions, such as heating and vacuum. The bimetallic nanoflowers provided a large surface area and excellent CAT-like activity, thereby enhancing electrochemical performance. Based on the principle of branched catalytic hairpin assembly, target miRNA could continuously trigger the assembly of branched junctions with ends composed of DNAzyme. The activated DNAzyme was able to cleave the hairpin substrate efficiently and release to capture more CeO2@Ag HNFs label. The process combining target-driven branched catalytic hairpin assembly and DNAzyme-assisted rolling cleavage were defined as TFRC signal amplification. This sensitive electrochemical biosensor exhibited good linear relationship of 1 fM − 1 nM with a detection limit down to 0.89 fM. The proposed method is expected to have a promising application in the miRNA-associated fundamental research and clinical diagnosis.

Development of an enhanced visual signal amplification assay for GSH detection with DNA-cleaving DNAzyme as a trigger

Glutathione (GSH) plays a significant role in human health. Considering that dietary intake of GSH is an important way to keep GSH levels in the body, it is particularly essential to quantitate the supplemental content of GSH. In this study, an enhanced visual signal amplification assay for GSH detection in food with DNA-cleaving DNAzyme as a trigger was developed. The trigger contained a Trigger-enzyme strand (Pb2+ dependent DNAzyme sequence) and a Trigger-substrate strand. In the absence of GSH, DNAzyme was formed with Pb2+ induction and Trigger-substrate strand was cleaved to release the initiator sequence. Initiator sequence could initiate RCA reaction and generate abundant G-quadruplex sequences. After G-quadruplex/hemin DNAzyme was formed in the presence of G-quadruplex sequences, a visual signal could be produced by catalyzing ABTS. In the presence of GSH, Pb2+ would combine with GSH and there would not be following visual signal production. Thus, GSH could be detected with a “turn-off” strategy and GSH concentration was negatively correlated with color change. Besides, Nb.BbvCI nicking endonuclease was introduced to improve the visual effect. The results could be qualitatively identified by the naked eye and quantitatively analyzed by spectrophotometry (Linearity range: 10 nM to 1 µM, LOD: 3.99 nM) with good selectivity and reliability. We also developed a smartphone-based visual analysis method to make the quantitative analysis independent on a spectrophotometer (Linearity range: 10 nM to 0.5 µM, LOD: 8.17 nM). We believe that this assay showed a good potential for rapid and sensitive detection of GSH in food.

Chaperone Copolymer Assisted G-Quadruplex-Based Signal Amplification Assay for Highly Sensitive Detection of VEGF.

Vascular endothelial growth factor (VEGF) is a critical biomarker in the angiogenesis of several cancers. Nowadays, novel approaches to rapid, sensitive, and reliable VEGF detection are urgently required for early cancer diagnosis. Cationic comb-type copolymer, poly(L-lysine)-graft-dextran (PLL-g-Dex) accelerates DNA hybridization and chain exchange reaction while stabilizing the DNA assembly structure. In this work, we examined the chaperone activity of PLL-g-Dex to assist G-quadruplex-based fluorescent DNA biosensors for sensitive detection of VEGF. This convenient and effective strategy is based on chitosan hydrogel, c-myc, Thioflavin T (ThT), VEGF aptamer, and its partially complementary strand. The results show that chaperone copolymer PLL-g-Dex significantly promotes the accumulation of G-quadruplex and assembles into G-wires, allowing an effective signal amplification. Using this method, the detection limit of VEGF was as low as 23 pM, better than many previous works on aptamer-based VEGF detection. This chaperone copolymer-assisted signal amplification strategy has potential applications in the highly sensitive detection of target proteins, even including viruses.

The PCR-HCR dual signal amplification strategy for ultrasensitive detection of Escherichia coli O157:H7 in milk

In this study, we established a novel polymerase chain reaction-hybridization chain reaction (PCR-HCR) dual signal amplification assay for sensitive detection of Escherichia coli O157:H7 in milk. In this assay, a blocker forward primer and a biotin reverse primer based on E. coli O157:H7 specific fliCh7 gene were specifically designed for PCR amplification resulting in a single-double stranded DNA with biotin (ss-dsDNA~biotin). These PCR products, ss-dsDNA~biotin, were captured by magnetic beads (MBs) decorated with streptavidin (SA) forming the ss-dsDNA~biotin+SA~MBs. The ssDNA on the PCR products opened the hairpin probe (H1 and H2) to trigger the HCR for secondary signal amplification. This is the first to use HCR to directly amplify PCR products through a novel blocker primer. With the help of two nucleic acid dual signal amplification techniques, PCR-HCR analysis can effectively improve the detection sensitivity and the limit of detection is 7.2 × 101 CFU/mL. In addition, this assay has been successfully used to detection target bacteria in skim milk spiked samples.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

Original signal amplification assay for N-Terminal pro-brain natriuretic peptide detection based on Bi2MoO6 photosensitive matrix

An original dual signal amplification immunoassay for N-Terminal pro-brain natriuretic peptide (NT-proBNP) detection is developed based on Au NPs and Zn doped CdS nanoparticles (Zn0.02Cd0.98S) co-sensitized Bi2MoO6 nanosheet photoelectrochemical (PEC) platform, the target is a good myocardial marker for diagnosing heart failure (HF). Bi2MoO6 as an outstanding photocatalysis material is successfully used in PEC analysis in this work, and the nanosheet structure provide a preponderance to capture superior Zn0.02Cd0.98S in order to achieve anticipant PEC response. The doping of Zn makes the energy band of CdS matched more compatible with Bi2MoO6, and improves the narrow band gap of CdS, making the surface plasma resonance (SPR) effect of Au NPs more significant, thus further improving the PEC response, as well as elevated the detection sensitivity of biological targets. The constructed PEC platform for NT-proBNP provides a wide detection range of 0.0001–1000 ng mL−1 and gives the minimum detection value of 0.037 pg mL−1. Great stability, high selectivity, and good reproducibility are also achieved. The proposed PEC immunoassay provides more possibilities for other protein ultra-sensitivity detection.

273 Multi-Target DNA Aberrations in Sporadic Colorectal Cancer Tissues Do Not Differ Between Younger and Older Patients

INTRODUCTION: Colorectal cancer (CRC) screening is broadly endorsed for individuals 50–75 years of age. Given recent increases in CRC incidence and mortality for patients &lt;50 years old, the American Cancer Society now recommends initiation of average-risk CRC screening at age 45 (Wolf AMD CA Cancer J Clin 2018). The performance of recommended tests, including the multi-target stool DNA (mt-sDNA; marketed as Cologuard™) test, has been minimally described in populations under 50 years old. To address this knowledge gap, we conducted a retrospective study of DNA markers (KRAS, BMP3, NDRG4) included in the mt-sDNA assay to quantify and compare tissue marker levels in sporadic CRC cases and normal colon controls from patients in 45–49 and 50–64 year-old age groups. Our primary hypothesis was that mt-sDNA marker levels would be statistically similar between cases in both age groups. METHODS: Patient samples were identified from a pathology tissue archive at Mayo Clinic, excluding those with inflammatory bowel disease or familial cancer syndromes. Formalin-fixed, paraffin-embedded tissues were reviewed by a GI pathologist for 45–49 year-old (n = 90) and 50–64 year-old (n = 96) CRC cases; normal colon samples (45–49, n = 76; 50–64, n = 92) were also included for comparison. DNA extracted from core punches was blinded and processed in a randomized order. Quantitative Allele specific Realtime Target and Signal amplification (QuARTS) assays were used to quantify KRAS mutations, and BMP3 and NDRG4 methylation. Marker level in each sample was reported as the QuARTS product of mutant KRAS, methylated BMP3 or NDRG4 relative to the ACTB product in each sample, reported as a percentage. At least 60 patients per group provided sufficient power to demonstrate differences in marker levels of ±25% between case groups and between control groups using the Wilcoxon Rank Sum test. RESULTS: CRC cases in the 45–49 vs 50–64 year-old groups did not differ significantly with respect to sex (P = 0.77) or race/ethnicity (P = 0.06); left-sided CRC was more common in the 45–49 vs 50–64 year-old group (87% vs. 72%; P = 0.02). None of the marker levels were statistically different between cases of the younger vs older groups or between controls of these groups (Figure 1). CONCLUSION: These data support the expectation of no age-related differences in CRC detection by mt-sDNA, as the relevant molecular markers, when they occur, are present at similar levels across age groups 45–49 and 50–64 years.

A T7 exonuclease assisted dual-cycle signal amplification assay of miRNA using nanospheres-enhanced fluorescence polarization

Based on streptavidin coated nanospheres and T7 exonuclease assisted dual-cycle signal amplification, we developed a novel sensitive fluorescence polarization detection method for miRNA. When target miRNA was present in the system, hairpin probe hybridized with miRNA, forming a double-stranded structure. The 5′ end of hairpin probe was then recognized and digested by T7 exonuclease, releasing the non-degraded single strand DNA fragments and miRNA. The released target miRNA could trigger the next cycle of hybridization and digestion, releasing more non-degraded fragments from hairpin probe. The fragments could hybridize with a signal probe (with carboxyfluorescein modification at 5′-end and biotin modification at 3′-end). The formed blunt 5′-end of signal probe was then recognized and degraded by T7 exonuclease, releasing the fragments and the fluorophore carboxyfluorescein. The next cycle of hybridization and digestion of signal probe was triggered by the released fragment at the same time. The free carboxyfluorescein cannot connect with streptavidin coated nanospheres which were used as the fluorescence polarization signal amplifier. So, there was a big change of fluorescence polarization signal after adding miRNA into the detection system, due to the different fluorescence polarization signal between nanospheres-captured intact signal probe and free carboxyfluorescein. The detection limit of this method is about 0.001 nM, and it has a good selectivity. In addition, it was also applicable to determine target miRNA in total miRNA extracts and compare the expression level of target miRNA in different cells. Consequently, the proposed method is expected to be used for the potential cancer diagnosis and the related biomedical research.

G-triplex based molecular beacon with duplex-specific nuclease amplification for the specific detection of microRNA.

High sequence homology among miRNA members challenges miRNA analysis. In this paper, we developed a simple and highly selective method based on a novel G-triplex molecular beacon (MBG3) and duplex-specific nuclease signal amplification (DSNSA) for miRNA detection. Herein, this MBG3 is a label-free molecular beacon with a special G-triplex probe. The excellent controllablity of the G-triplex probe allowed our MBG3 to have a short stem, which protected it from DSN digestion. Therefore, DSN cleavage induced false positive signals in most DSN signal amplification strategies have been minimised efficiently without special modifications. Importantly, the improved recognition ability of MBG3, together with the sensitive substrate selectivity of DSN, makes our novel method suitable for miRNA detection with high selectivity. The signal response of similar miRNA sequences with one-base difference has been reduced from 37% to 8% compared to the traditional linear ssDNA probe-DSN-based method.

Fluorescence signal amplification assay for the detection of B. melitensis 16M, based on peptide-mediated magnetic separation technology and a AuNP-mediated bio-barcode assembled by quantum dot technology

Members of the Brucella spp. are facultative intracellular bacteria that can cause global brucellosis, a zoonotic disease. Herein, a novel fluorescence signal amplification (FSA) method for the rapid detection of B. melitensis 16M was developed based on peptide-mediated magnetic separation (PMS) technology and Au nanoparticle (AuNP)-mediated bio-barcode assay technology assembled by quantum dots (QDs). The PMS technology was used to specifically capture and isolate B. melitensis 16M from food. The immunomagnetic bead-B. melitensis 16M bioconjugates (IMBs-B. melitensis 16M) were then identified by IgY on the surface of AuNPs and the oligonucleotide chains on the surface of the gold nanoparticles were hybridized with bio-barcodes assembled by quantum dots (QD-probe2). The IMB/B. melitensis 16M/IgY-AuNP-probe1/QD-probe2 bioconjugates were concentrated by magnetic separation. Therefore, as the concentration of B. melitensis 16M in the sample increased, the unbound QD-probe2 in the supernatant reduced, and the B. melitensis 16M in the sample could be indirectly measured by detecting the fluorescence in the supernatant. This FSA method can detect B. melitensis 16M concentration in the range of 10 to 106 cfu ml-1 without pre-enrichment, and the limit of detection (LOD) is as low as 10 cfu ml-1 with high specificity. Furthermore, the proposed method for the detection of B. melitensis 16M has a LOD of 1.07 × 102 cfu ml-1 and a linear range from 102 to 107 cfu ml-1 in milk, and a LOD of 1.72 × 102 cfu ml-1, and a linear range from 102 to 106 cfu ml-1 in lamb leach. In addition, this method takes less than 3 h to perform. Thus, the assay that was developed in this study shows promise for rapid, sensitive, and specific detection of B. melitensis 16M.

Ternary Electrochemiluminescence Nanostructure of Au Nanoclusters as a Highly Efficient Signal Label for Ultrasensitive Detection of Cancer Biomarkers.

Herein, the Au nanoclusters (Au NCs) functionalized ternary nanostructure with significant electrochemiluminescence (ECL) emission was first proposed to fabricate an ultrasensitive immunosensor for carcinoembryonic antigen (CEA) detection, which was established by combining bovine serum albumin (BSA) templated Au NCs as the luminophor, tris(3-aminoethyl)amine (TAEA) as the coreactant, and Pd@CuO nanomaterial as the coreaction accelerator via covalent attachment in a nanostructure. Through the dual self-catalysis including intramolecular coreaction between TAEA and Au NCs, and intramolecular coreaction acceleration from Pd@CuO to TAEA, the Au NCs-TAEA-Pd@CuO achieved excellent ECL performance so that the detection limit of the immunosensor for measuring CEA antigen was down to 16 fg/mL in the absence of any additional signal amplification assay. Moreover, the method for preparing ECL nanostructure-based metal NCs should be devoted to the development of highly efficient ECL signal label, outlining a significant scheme toward biological testing and clinical diagnosis.

Ultrasensitive electrochemiluminescence detection of Staphylococcus aureus via enzyme-free branched DNA signal amplification probe

As a main cause of foodborne diseases, pathogenic bacteria have threatened the health and well-being of human communities. There is a need of fastness, accuracy and sensitivity in the method of detecting pathogenic bacteria. Classical signal amplification assays usually employ enzymes as biocatalysts to generate amplified signals, but the strict experimental conditions and complicated instruments restrict their application. In this work, we demonstrated an enzyme-free branched DNA (bDNA)-based signal amplification electrochemiluminescence (ECL) assay for ultrasensitive detection of pathogenic bacteria. Firstly, the capture probes and the amplification probes group were carefully designed by our research group. The detecting ECL signal of Staphylococcus aureus (S. aureus) was amplified by bDNA technique through the layer-by-layer signal amplification. The sensitivity was greatly improved by the use of multiple Ru(bpy)32+ (TBR)-labeled ECL probes. Secondly, the whole process of the detection was carried out in the absence of enzyme, without the need to control the reaction conditions strictly. Thirdly, the designed amplification probes group could be used for the analysis of other pathogenic bacteria, virus, tumor markers, biomarkers, etc. For the detection of S. aureus, the limit of detection (LOD) of the method was 2 pM for standard DNA, with the linear range from 20 pM to 100 nM. Last but not least, the LOD of the S. aureus asymmetric PCR products was 5 pM, with the linear range from 10 pM to 50 nM. The sensitivity was 1–2 orders in magnitude higher than that of the common detection assays.

A DNA-linker-DNA bifunctional probe for simultaneous SERS detection of miRNAs via symmetric signal amplification.

In this work, using a dual functional DNA-linker-DNA (DLD) probe, a new concept of a symmetric signal amplification (SSA) reaction is introduced to simultaneously analyze miRNAs. By coupling the surface-enhanced Raman scattering (SERS) technology with symmetric amplification modes, this flexible biosensing system exhibits high sensitivity and specificity.

Abstract 712: Detection of lung cancer by assay of novel methylated DNA markers in plasma

Abstract Purpose: Lung cancer is the leading cause of cancer deaths worldwide. Most present symptomatically at late stage with high lethality. Early detection reduces mortality but accurate and readily accessible tools for population screening are limited. By whole methylome sequencing, we have identified novel methylated DNA markers (MDMs) for lung cancer in tissue (Giakoumopoulos et al. ASCO 2016). Using top candidate MDMs in the present study, we now explore their clinical accuracy for lung cancer detection when assayed from plasma. Experimental Procedures: Archival plasmas from two independent study groups were tested in blinded fashion. Lung cancer cases and controls (apparently healthy smokers) for each group were balanced on age and sex (Group 1: 64 cases, 231 controls; Group 2: 23 cases, 80 controls). Using multiplex PCR followed by QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) assay, a post-bisulfite quantification of MDMs on DNA extracted from plasma was performed. We selected 31 MDM candidates for initial evaluation in Group 1 (1 ml plasma/patient); top individual MDMs were subsequently tested in Group 2 to identify optimal MDM panels for lung cancer detection (2 ml/patient). Results: From Group 1 analyses, 13 high performance MDMs were selected for further testing (CYP26C1, SOBP, SUCLG2, SHOX2, ZDHHC1, NFIX, FLJ45983, HOXA9, B3GALT6, ZNF781, SP9, BARX1, EMX1) with individual areas under the receiver operator curve (AUCs) ranging from 0.593 to 0.939. Discrimination by individual MDMs was corroborated in Group 2 in which data was analyzed using two methods: a logistic regression fit and a regression partition tree approach. The logistic fit model identified a 4-marker panel (ZNF781, BARX1, EMX1, and SOBP) with an AUC of 0.96 and an overall sensitivity of 91% and 90% specificity. Analysis of the data using a regression partition tree approach identified 4 markers (ZNF781, BARX1, EMX1, and HOXA9) with AUC of 0.93 and an overall sensitivity of 96% and specificity of 94%. For both approaches, B3GALT6 was used as a standardizing marker of total DNA input. Conclusion: A panel of MDMs assayed in plasma achieved high sensitivity and specificity for all types of lung cancer. Further clinical evaluation and validation of this promising panel in larger patient groups are clearly indicated. Citation Format: Hatim T. Allawi, Maria Giakoumopoulos, Evan Flietner, Austin Oliphant, Carla Volkmann, Brian Aizenstein, Tamara Sander, Drew Eckmayer, Ashley Poenitzsch Strong, Melissa Gray, Barry Berger, Tracy Yab, William Taylor, Douglas Mahoney, John B. Kisiel, David E. Midthun, David A. Ahlquist, Graham P. Lidgard. Detection of lung cancer by assay of novel methylated DNA markers in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 712. doi:10.1158/1538-7445.AM2017-712

The new frontier of diagnostics: Molecular assays and their role in infection prevention and control.

Recent advances in technology over the last decade have propelled the microbiology laboratory into a pivotal role in infection prevention and control. The rapid adaptation of molecular technologies to the field of clinical microbiology now greatly influences infectious disease management and significantly impacts infection control practices. This review discusses recent developments in molecular techniques in the diagnosis of infectious diseases. It describes the basic concepts of molecular assays, discusses their advantages and limitations, and characterizes currently available commercial assays with respect to cost, interpretive requirements, and clinical utility.