Single Copy Sensitivity Research Articles

Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection.

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

A cost-effective and field-deployable sensing system for chip-integrated detection of bacteria with the naked eye

To tailor the broadly applied polymerase chain reaction (PCR) technology to point-of-care settings, an intuitive, time-saving, and cost-effective method to determine the DNA amplification results has significant practical potential. Herein we present an economical and field-deployable sensing system that enables naked-eye readout of PCR results. The proposed system is powered by an amplification enhancer to greatly enhance the positive-to-background ratio and a palm-sized readout device to assist on-site applications. High specificity, as well as single-copy sensitivity, was achieved in detecting Salmonella enterica and other bacterial pathogens. Using the proposed system, we identified the pathogenic bacterial strains in artificially contaminated food samples easily, quickly, and reliably. Also crucial for point-of-care testing, this naked-eye readout system applies to microfluidic platforms with the sample volumes scaled down to 1 μL. The time savings, portability, and cost-effectiveness of the established sensing system make it transformative for on-chip bacteria detection in point-of-care and field settings, especially in resource-limited areas.

Comparative sensitivity of automated (Abbott M2000) and manual plasma HIV-1 RNA PCR assays for the detection of persistent viremia after long-term antiretroviral therapy

BackgroundThe ability of automated, FDA-cleared plasma HIV-1 RNA assays to detect low-level viremia, compared to manual, highly sensitive research-only methods, is not well-defined. We therefore tested paired plasma samples from people with HIV-1 (PWH) on long-term antiretroviral therapy (ART) with both the Abbott M2000 RealTime HIV-1 Viral Load assay (Abbott) and a quantitative reverse transcriptase (RT)-initiated PCR assay that has a reported 95% detection limit of 1 HIV-1 RNA copy/ml (single copy assay, SCA). MethodsPlasma samples from 309 participants in the AIDS Clinical Trials Group study A5321 were tested by both Abbott and SCA. Participants were mostly men (82%). All were on stable ART for a median of 7 years with HIV-1 RNA <40 copies/mL by Abbott. Pooled plasma from each donor was divided and tested. Abbott results were reported as target detected <40 copies/mL but not quantifiable (target detected <40) or target not detected (TND), and SCA results were classified as HIV-1 RNA detected or not detected. ResultsBy Abbott, 17% (51/309) of sample results were target detected <40, whereas 83% (258/309) were TND. Of the samples that were target detected <40 by Abbott, 73% (37/51) had HIV-1 RNA detected by SCA. By contrast, 43% of samples that were TND by Abbott (110/258) had HIV-1 RNA detected by SCA (p < 0.001). ConclusionPlasma samples from PWH with HIV-1 RNA detected but <40 copies/ml by the automated Abbott M2000 assay are likely (73% of 51 samples) to have HIV-1 RNA detected by an optimized manual assay with single copy sensitivity. An Abbott HIV-1 RNA result of target not detected did not exclude low-level viremia: 43% of 258 samples had HIV-1 RNA detected by the single copy assay. These findings indicate that the Abbott M2000 assay cannot exclude the persistence of viremia on ART and thus may have less utility, compared to a manual single copy assay, for assessing the impact of experimental interventions designed to eliminate low-level viremia as a step towards achieving ART-free HIV-1 remission.

Amine-Functionalized Quantum Dots as a Universal Fluorescent Nanoprobe for a One-Step Loop-Mediated Isothermal Amplification Assay with Single-Copy Sensitivity.

Loop-mediated isothermal amplification (LAMP) has received considerable attention for decentralized (point-of-care and on-site) nucleic acid testing in view of its simple temperature control (60-65 °C) and short assay time (15-60 min). There remains a challenge in its wide adoption and acceptance due to the limitations of the existing amplification result reporter probes, e.g., photobleaching of organic fluorophore and reduced sensitivity of the pH-sensitive colorimetric dye. Herein, we demonstrate CdSeS/ZnS quantum dots (semiconductor fluorescent nanocrystals with superior photostability than organic fluorophore) with surface modification of cysteamine (amine-QDs) as a new reporter probe for LAMP that enabled single-copy sensitivity (limit of detection of 83 zM; 20 μL reaction volume). For a negative LAMP sample (absence of target sequence), positively charged amine-QDs remained dispersed due to interparticle electrostatic repulsion. While for a positive LAMP sample (presence of target sequence), amine-QDs became precipitated. The characterization data showed that amine-QDs were embedded in magnesium pyrophosphate crystals (generated during positive LAMP), thus leading to their coprecipitation. This amine-QD-based one-step LAMP assay advances the field of QD-based nucleic acid amplification assays in two aspects: (1) compatibility─one-step amplification and detection (versus separation of amplification and detection steps); and (2) universality─the same amine-QDs for different target sequences (versus different oligonucleotide-modified QDs for different target sequences).

Detection of Four Porcine Enteric Coronaviruses Using CRISPR-Cas12a Combined with Multiplex Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay.

Porcine enteric coronaviruses have caused immense economic losses to the global pig industry, and pose a potential risk for cross-species transmission. The clinical symptoms of the porcine enteric coronaviruses (CoVs) are similar, making it difficult to distinguish between the specific pathogens by symptoms alone. Here, a multiplex nucleic acid detection platform based on CRISPR/Cas12a and multiplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of four diarrhea CoVs: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). With this strategy, we realized a visual colorimetric readout visible to the naked eye without specialized instrumentation by using a ROX-labeled single-stranded DNA-fluorescence-quenched (ssDNA-FQ) reporter. Our method achieved single-copy sensitivity with no cross-reactivity in the identification and detection of the target viruses. In addition, we successfully detected these four enteric CoVs from RNA of clinical samples. Thus, we established a rapid, sensitive, and on-site multiplex molecular differential diagnosis technology for porcine enteric CoVs.

CRISPR-microfluidic array for single-copy DNA mini barcoding and rapid field species identification

DNA barcode identification has wide applications in taxonomy, molecular phylogenetics, biodiversity science and species identification. This work developed a CRISPR-microfluidic array for rapid identification of DNA barcodes. This assay has successfully distinguish various types of Datura L. with single-copy sensitivity and rapidity. The detection system achieved 100% consistency with the morphological identification in detecting real-field seeds, holding great potential in the field of DNA barcoding and species recognition.

A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis

ABSTRACT As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

A critical review: Recent advances in “digital” biomolecule detection with single copy sensitivity

Detection of a single biomolecule, ranging from nucleic acids, proteins, viruses to bacteria, is of paramount importance in various fields including biology, environment, food and agriculture industry, public health, and medicine. With the understanding of the biological functions of these biomolecules (or bioparticles) and their impacts on public health, environmental pollution, and food safety, advanced detection techniques are unprecedentedly demanded for their early and/or sensitive detection. In this critical review, a series of elegant research about digital detection of biomolecules with potential single copy sensitivity is reviewed and summarized with the focus on the design principle and the innovation of how to accomplish the “digital” detection concept. Starting with a brief introduction on the importance of digital detection, recent advances in “digital” biomolecule detection with single copy sensitivity are grouped and discussed based on the difference of signal reporting systems, including surrogate signal development for “digital” detection, direct visualization for “digital” detection, and nucleic acid amplification enabled “digital” detection. Interdisciplinary combination and integration of different cutting-edge techniques are also discussed with details. The review is closed with the conclusion and future trends.

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual integrase-targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.

CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis

ABSTRACT Rapid and simple-to-use diagnostic methods for tuberculosis are urgently needed. Recent development has unveiled the diagnostic power of the CRISPR system in the detection of viral infections. However, its potential use in detecting the Mycobacterium tuberculosis complex (MTB) remained unexplored. We developed a rapid CRISPR-based assay for TB detection and conducted a retrospective cohort study of 179 patients to evaluate the CRISPR-MTB test for identifying MTB in various forms of direct clinical samples. Its diagnostic performance was compared, in parallel with culture and the GeneXpert MTB/RIF assay (Xpert). The CRISPR-MTB test is highly sensitive with a near single-copy sensitivity, demands less sample input and offers shorter turnaround time than Xpert. When evaluated in the clinical cohort of both pulmonary and extra-pulmonary tuberculosis, the CRISPR-MTB test exhibited an overall improved sensitivity over both culture (79% vs 33%) and Xpert (79% vs 66%), without comprise in specificity (62/63, 98%). The CRISPR-MTB test exhibits an improved overall diagnostic performance over culture and Xpert across a variety of sample types, and offers great potential as a new diagnostic technique for both pulmonary and extra-pulmonary tuberculosis.

Abstract 4534: Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations

Abstract Background: Accurate detection of driver mutations in cancer patients is vital for targeted therapy. Compared to tissue biopsy, “liquid biopsy” offers a non-invasive and more systemic approach to identify tumor mutations by assessing circulating tumor DNA (ctDNA) released from tumor cells into peripheral blood. We have developed TargetSelectorTM Real-Time PCR based assays to detect low frequency mutant alleles in ctDNA. The TargetSelectorTM assay uses a proprietary blocker to suppress amplification of excess WT alleles released from normal cells, while allowing specific amplification of mutants. Here we focus on five important targets: EGFR (Del19, L858, and T790), BRAF (V600), and KRAS (G12/G13), which are relevant to lung cancer, melanoma, and colorectal cancer. Methods: The TargetSelectorTM assay applies a specific blocker to cover variants on a short stretch of target DNA (up to 10 bp for nucleotide variants). For example, one KRAS exon 2 blocker covers all variants on both G12 and G13 positions. The TargetSelectorTM assays were first validated with cancer cell line DNA carrying mutation targets on QuantStudio 5 Real-Time PCR instruments (QS5). Sanger Sequencing was subsequently performed to confirm the mutation. Analytical validation was conducted by 3 independent operators using 5 instruments across 5 days in our CAP/CLIA certified laboratory. For ctDNA testing, whole blood samples were collected in CEE-SureTM Blood Collection tubes and DNA extraction from plasma was performed on the QIAsymphony. Results: In total, we tested 3086 samples for EGFR, BRAF and KRAS TargetSelectorTM ctDNA assays, with EGFR WT assay as the background reference. All five ctDNA assays showed &amp;gt;99% analytical sensitivity and &amp;gt;99% analytical specificity. Based on practical and theoretical estimates, each ctDNA assay demonstrated single mutant copy detection sensitivity. In the presence of 14,000 copies of WT background, the sensitivity of our ctDNA assays are: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. The inter-assay and intra-assay analyses showed r2 &amp;gt;0.94, suggesting a consistent performance among operational variables. Samples tested from 20 healthy donors (100 tests in total) showed clinical specificity &amp;gt;99%. In the concordance study of 13 clinical samples (31 tests in total) between QS5 and ABI 7900HT platforms, TargetSelectorTM ctDNA assays with the QS5 identified the same plus additional mutations compared to the 7900HT. Conclusions: TargetSelectorTM ctDNA assays were validated both analytically and clinically, showing single mutant copy detection and sensitivity at 0.02% or better in a background of excess WT DNA. Implementation of the QS5 qPCR platform into our TargetSelectorTM ctDNA assays leads to a higher sensitivity and faster turnaround time. These factors enable sensitive and efficient testing crucial for guiding treatment decisions and patient care. Citation Format: Shan-Fu Wu, Timothy T. Lu, Anh Pham, Jeffrey Chen, Tony Daher, Errin Samuelsz, Manisha Patel, Veena M. Singh, Lyle J. Arnold, Jason C. Poole. Validation of highly sensitive TargetSelectorTM ctDNA assays for EGFR, BRAF, and KRAS mutations [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 4534.

Abstract 4666: DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA

Abstract Background: Current approaches for the assessment of methylation, such as methylation-specific PCR (MSP) and next-generation bisulfite sequencing (BS-Seq) are fundamentally limited in their ability to detect and assess heterogeneous methylation patterns (epialleles) in ultra-rare (&amp;lt;0.1%) DNA. These limitations critically compromise diagnostic utility and render them ill suited for many emerging applications in cancer diagnostics, such as the analysis of methylation heterogeneity in cell-free DNA (cfDNA) and rare cell populations. We recently addressed the need for a low cost alternative to the assessment of methylation of ultra-rare DNA with the development of DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of DNA at single copy sensitivity and single-CpG-site resolution. Here, we seek to demonstrate the advantages of the DREAMing method over conventional approaches to methylation assessment. Methods: We expand upon the underlying theory of DREAMing and provide guidelines for the development of single-copy sensitive DREAMing assays. We further elucidate methods for tailoring DREAMing assays to samples of interest and compare the performance of these assays to commonly employed techniques including quantitative MSP (qMSP) and BS-Seq. Results: Development of single-copy sensitive DREAMing assays for a number of loci associated with classic tumor-specific methylation such as CHFR and RASSF1A as well as a candidate pan-cancer locus are reported. These assays are then used to analyze methylation in cfDNA derived from the plasma of cancer-positive and healthy patients. DREAM analysis reveals that DREAMing can readily detect over an order of magnitude more epialleles when directly compared to qMSP and BS-Seq assays of the same locus. Some of the challenges associated with distinguishing potential tumor-specific aberrant methylation from background methylation are then discussed and proposed solutions are demonstrated. Lastly, methods for optimizing DREAMing assays for specific sample types are discussed. Conclusions: DREAMing is a recently introduced method for the assessment of locus-specific methylation in samples containing ultra rare target DNA. Its low cost and simplicity coupled with the ability to provide enhanced, single-copy detection of heterogeneous methylation make DREAMing an attractive option over traditional techniques for demanding specimens such as cfDNA and rare cell populations. DREAMing has potential utility in the evaluation of DNA methylation dynamics in cell populations, prenatal testing, as well as clear use in early cancer diagnostic, companion diagnostic and predictive applications. Citation Format: Thomas R. Pisanic, Pornpat Athamanolap, Brendan F. Miller, Vincent Wu, Laura Elnitski, Tza-Huei Wang. DREAMing as a simple and low cost alternative for the assessment of methylation in ultra rare DNA [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 4666. doi:10.1158/1538-7445.AM2017-4666

Non-fluorescent quantification of single mRNA with transient absorption microscopy.

Single molecule detection is confounded by the background signals from the biological environment, such as autofluorescence, Rayleigh scattering, or turbidity in cells and tissues. In this article, we report on the utilization of gold nanoparticles (AuNPs) as an orthogonal probe for non-fluorescence detection of single molecules with a transient absorption microscopy (TAM). The developed system and concepts were validated by quantitative evaluation of human epidermal receptor 2 (Her2) mRNA in cancer cells and tissues at single copy sensitivity. Results from TAM suggest that the average number of Her2 copies in SK-BR-3 and MCF-7 breast cancer cells is 203.19 ± 80.48, and 11.29 ± 4.47, respectively. Furthermore, TAM offers excellent signal-to-noise ratio in detecting mRNA in clinical tissues, indicating a significantly higher expression of Her2 genes in breast cancer tissues than that of normal tissues. Our single cell quantification TAM strategy was validated with a fluorescence in situ hybridization approach. Our demonstration shows that TAM has the potential to provide a new dimension in biomarker quantification at single molecule sensitivity in turbid biological environments providing a strong basis for clinical monitoring.

Abstract 30: Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response

Abstract Background: Acquisition of the EGFR T790M resistance mutation is a hallmark of disease progression in patients with metastatic EGFR mutant lung adenocarcinoma treated with first generation anti-EGFR inhibitors. Utilizing a single copy sensitivity mutation detection platform and implementing daily collection of urine samples, we sought to demonstrate the feasibility of detecting EGFR mutations in urinary ctDNA and understand mechanisms of resistance to targeted therapies in patients with EGFR-mutated lung adenocarcinoma. Methods: In a biomarker study of 100 patients with EGFR-mutant metastatic lung adenocarcinoma (34 patients enrolled), urine was collected at either daily or monthly time points up to 4 months prior to radiologic detection of progression on erlotinib, and at multiple time points post-progression on next line therapy. Urinary ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Quantitative analysis of EGFR activating mutations and T790M resistance mutation was performed using blocker technology and PCR enrichment coupled with NGS detection (MiSeq). Urine was collected daily after the initiation of second line anti-EGFR therapy in 10 patients. Early pharmacodynamic events that occur within the first hours to days of anti-EGFR therapy were further studied by quantitating ctDNA for EGFR exon 19deletions, L858R, and T790M. Results: Interim analysis was conducted on 34 patients receiving first line anti-EGFR therapy with erlotinib; twenty-two of 34 patients demonstrated radiographic progression. Analysis of longitudinal samples revealed that the EGFR T790M mutation was detected in the urine specimens of 15 out of 22 (68%) patients on erlotinib. All 10 patients who were positive for T790M mutation by tissue were also positive by urine. Urine testing identified five additional T790M-positive patients who had a high clinical suspicion of T790M progressive disease. Three of these patients were tissue negative but both plasma and urine positive for T790M. EGFR T790M was detected up to 15 weeks prior to radiolographic detection of progression on first line erlotinib. Early peaks in ctDNA on days 1-4 correlated with tumor lysis. An observed sustained decrease in mutational levels after week 1 of therapy confirms the cytostatic effect of the tyrosine kinase inhibitor (TKI). The size of the initial peaks in ctDNA for EGFR exon 19deletions, L858R, and T790M correlated with CT radiographic response after two cycles of therapy. Conclusion: We demonstrate that the T790M mutation can be successfully detected in urinary ctDNA months before progression on anti-EGFR TKIs. Urinary ctDNA testing identifies additional patients who potentially are eligible for anti-T790M treatment. Initial results from 10 patients demonstrated that kinetic changes in EGFR ctDNA mutational load after drug adminstration can be used with pharmacokinetic data to better understand dyanmic changes in tumor biology, drug bioavailability, and assist in early drug development. The clinical utility of daily kinetic monitoring of ctDNA in urine after drug adminstration is being further validated in a larger study. Citation Format: Hatim Husain, Karena Kosco, Saege Hancock, Errin Samuelsz, Shiloh Guerrero, Brian Woodward, Cecile Rose Vibat, Vlada Melnikova, Mark Erlander, Scott Lippman, Razelle Kurzrock. Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 30.

Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy.

Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ.

Abstract 5238: Methodology for single copy detection and quantitative monitoring of clinically actionable circulating tumor DNA mutations in urine from cancer patients

Abstract Background: Non-invasive detection and monitoring of circulating tumor DNA (ctDNA) mutations for personalized treatment of cancer patients can be realized by combining the practical advantages of urine as a ctDNA sample source with high throughput of next-generation sequencing (NGS). Methods: Our platform couples an extraction process capable of isolating ctDNA from the entire void volume of a urine sample (∼100ml) with an ultra-sensitive NGS-integrated mutation enrichment method with single copy detection sensitivity. Assays have been developed and validated to interrogate clinically actionable mutations/deletions in the KRAS, BRAF and EGFR (Exons 19, 20, 21) oncogenes in both urine and plasma samples. For mutation detection with high sensitivity, a novel allele-specific competitive cycling (ASCC) method was used prior to NGS to amplify ultra-short target DNA (31-45 bp) using kinetically-favorable binding conditions for a wild type (WT) blocking oligonucleotide. Enriched amplicons were sequenced and a proprietary algorithm was used to quantify the mutant ctDNA input level in analytical and clinical samples. Results: An extraction method optimized for enrichment of fragmented urine ctDNA enabled isolation of large amounts of DNA (mean ∼2 μg) from advanced stage patients with different cancer types. Analytical performance characterization demonstrated sensitivity of 0.0047 - 0.01% mutant copies in WT/mutant DNA blends. To show that our assays have a true single copy detection threshold, we used DNA blends with defined mutant spike-in levels of 2 - 60 copies distributed over 20 wells (to obtain 0 - 3 mutant copies/well). The observed distribution of positive and negative hits matched the theoretical hit rate of an ideal Poisson distribution for these replicates, confirming single copy sensitivity of our assays. Using the KRAS ASCC assay, 1 to 17,555 mutant fragments were detected per 1 mL of urine collected from KRAS tissue biopsy-positive advanced cancer patients. Analysis of serial patient-matched urine and plasma longitudinal samples from KRAS, BRAF, or EGFR tissue positive patients demonstrated a high level of concordance between urine and plasma samples and feasibility of monitoring mutation load in a variety of clinical settings, including monitoring post-surgery and responsiveness to targeted therapy or chemotherapy. Conclusion: The analytical characterization and clinical feasibility studies demonstrate that this methodology can successfully detect and quantitate mutational load in urinary ctDNA, thus enabling for the dynamic monitoring of therapy response, drug resistance, and minimal residual disease in cancer patients from a truly non-invasive sample. Citation Format: Karena Kosco, Jason C. Poole, Saege Hancock, Errin Samuelsz, Timothy T. Lu, Erin Clark, Latifa Hassaine, Shiloh Guerrero, Cecile Rose T. Vibat, Vlada Melnikova, Mark G. Erlander. Methodology for single copy detection and quantitative monitoring of clinically actionable circulating tumor DNA mutations in urine from cancer patients. [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 5238. doi:10.1158/1538-7445.AM2015-5238

Second harmonic super-resolution microscopy for quantification of mRNA at single copy sensitivity.

Cell-specific information on the quantity and localization of key mRNAs at single copy sensitivity in single cells is critical for evaluating basic cellular process, disease risk, and efficacy of therapy. Quantification of overexpressed mRNAs beyond the diffraction limit is constrained by the optical property of the probes and microscopy techniques. In this report, nanosized barium titanium oxide (BaTiO3, BTO) crystals were utilized as probes for mRNA quantification by a second harmonic super-resolution microscopy (SHaSM). The SHaSM was able to detect a single copy of the human epidermal growth factor receptor 2 (Her2) mRNA at a resolution of 55.6 nm with the ability to resolve multiple mRNA copies in a diffraction-limited spot. Her2 mRNA per cell was counted in SK-BR-3, MCF-7, and HeLa cell lines as 595 ± 79.1, 38.9 ± 8.26, and 1.5 ± 2.8, respectively. Our single-cell quantification results were validated with the fluorescence in situ hybridization studies and quantitative PCR, showing better specificity and selectivity over current single-molecule approaches for transcript detection. The SHaSM is expected to have an upper limit of resolving ∼104 transcripts in a single cell with the ability to monitor intracellular transcriptional dynamics at video rate. The developed approach has strong potential in clinical research and in the early diagnosis of life-threatening diseases such as cancer.

Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples

Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.

Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A/H1, A/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity

BackgroundRapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR. ObjectiveTo develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction. Study designWe designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens. ResultsOptimized M-LAMP was rapid with a mean amplification time of 12min (compared with 90–120min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A/H1 and A/H3 from influenza B by Tm. M-LAMP detected 26/28 influenza A/H1, 27/27 influenza A/H3 and 39/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92/94) and 100% (108/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result. ConclusionsM-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30min.

Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

We present an approach for fluorescent insitu detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent insitu hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive insitu hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.