Single-molecule Amplification Research Articles

Clinical-Molecular characteristics and Post-Translational modifications of colorectal cancer in north China: Implications for future targeted therapies

This study delineated the elucidate molecular changes and their post-translational modifications (PTMs) in heterogenetic colorectal cancer (CRC) for a deeper understanding of the CRC pathophysiology and identifying potential therapeutic targets. In this retrospective study, the profiles of 13 hot spot gene mutations were analyzed and the microsatellite instability (MSI) status was determined.Employing the Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) assay, the clinical-pathological features of CRC were characterized in 249 Chinese patients. PTMs were quantified online.Among the patients with CRC, the mutation frequencies of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC genes were 47.8%, 3.6%, 4.8%, 13.7%, 55.8%, and 36.9%, respectively. The proportion of MSI-high (MSI-H) was 7.8%.Subsequent multiple logistic regression analysis showed significant associations including a link between lung metastasis and KRAS mutation, between liver metastasis and lymph node metastasis, between MSI-H and early-onset CRC (EOCRC) and KRAS mutation, between right-sided colon cancer and peritoneal metastasis, and between PIK3CA mutation and PTEN mutation. Patients with KRAS mutation presented with MSI-H, lung metastasis, and PIK3CA mutation. MSI-H, BRAF mutation, and PTEN mutation were more frequent in EOCRC. Phosphorylation and ubiquitylation were found in KRAS, BRAF, PTEN, and SMAD4; SUMOylation and ubiquitylation were observed in HRAS and NRAS; while phosphorylation was obvious in APC, P53, and MLH1. Notably, Phosphorylation and ubiquitylation were the two most common PTMs. The biological characteristics of CRC in Chinese patients have some unique clinical features, which can be explained by the genetic mutation profile, correlations among gene mutations and clinical characteristics. These distinctions set the Chinese patient population apart from their Western counterparts.

Fast Thermocycling in Custom Microfluidic Cartridge for Rapid Single-Molecule Droplet PCR.

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.

Single-molecule RNA capture-assisted droplet digital loop-mediated isothermal amplification for ultrasensitive and rapid detection of infectious pathogens

To minimize and control the transmission of infectious diseases, a sensitive, accurate, rapid, and robust assay strategy for application on-site screening is critical. Here, we report single-molecule RNA capture-assisted digital RT-LAMP (SCADL) for point-of-care testing of infectious diseases. Target RNA was captured and enriched by specific capture probes and oligonucleotide probes conjugated to magnetic beads, replacing laborious RNA extraction. Droplet generation, amplification, and the recording of results are all integrated on a microfluidic chip. In assaying commercial standard samples, quantitative results precisely corresponded to the actual concentration of samples. This method provides a limit of detection of 10 copies mL−1 for the N gene within 1 h, greatly reducing the need for skilled personnel and precision instruments. The ultrasensitivity, specificity, portability, rapidity and user-friendliness make SCADL a competitive candidate for the on-site screening of infectious diseases.

A novel method of digital ELISA using single molecule amplification enables sub-attomolar detection limits for blood-based proteomics and biomarker measurements

Abstract The application of measuring low-abundance protein biomarkers in blood (serum or plasma) can often present challenges due to limits of sensitivity or precision. Digital proteomics technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional immunoassay method. However, current digital ELISA technologies can lack sufficient sensitivity to quantitatively measure many low abundance proteins in blood samples. We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule amplification with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (5000 vs. 500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency). Using this approach, we characterize limit of detection (LOD) of of 10–100X more sensitive than the standard digital ELISA. A digital ELISA with improved sensitivity was used to measure cytokines in serum and plasma samples with values consistently above the assay lower limits of quantitation (LLoQ). The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity. The novel approach to multiplex single molecule proteomics with attomolar sensitivity provides the potential to accelerate insights in proteomics biomarkers research, clinical applications and diagnostics.

Multi-omics integrated circulating cell-free DNA genomic signatures enhanced the diagnostic performance of early-stage lung cancer and postoperative minimal residual disease

Liquid biopsy is a promising non-invasive alternative for cancer screening and minimal residual disease (MRD) detection, although there are some concerns regarding its clinical applications. We aimed to develop an accurate detection platform based on liquid biopsy for both cancer screening and MRD detection in patients with lung cancer (LC), which is also applicable to clinical use. We applied a modified whole-genome sequencing (WGS) -based High-performance Infrastructure For MultIomics (HIFI) method for LC screening and postoperative MRD detection by combining the hyper-co-methylated read approach and the circulating single-molecule amplification and resequencing technology (cSMART2.0). For early screening of LC, the LC score model was constructed using the support vector machine, which showed sensitivity (51.8%) at high specificity (96.3%) and achieved an AUC of 0.912 in the validation set prospectively enrolled from multiple centers. The screening model achieved detection efficiency with an AUC of 0.906 in patients with lung adenocarcinoma and outperformed other clinical models in solid nodule cohort. When applied the HIFI model to real social population, a negative predictive value (NPV) of 99.92% was achieved in Chinese population. Additionally, the MRD detection rate improved significantly by combining results from WGS and cSMART2.0, with sensitivity of 73.7% at specificity of 97.3%. In conclusion, the HIFI method is promising for diagnosis and postoperative monitoring of LC. This study was supported by CAMS Innovation Fund for Medical Sciences, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Beijing Natural Science Foundation and Peking University People's Hospital.

Ultrafast and absolute quantification of SARS-CoV-2 on food using hydrogel RT-LAMP without pre-lysis.

With rapid growing of environmental contact infection, more and more attentions are focused on the precise and absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus on cold chain foods via point-of-care test (POCT). In this work, we propose a hydrogel-mediated reverse transcription loop-mediated isothermal amplification (RT-LAMP) for ultrafast and absolute quantification of SARS-CoV-2. Cross-linked hydrogel offers opportunities for digital single molecule amplification in nanoconfined spaces, facilitating the virus lysis, RNA reverse transcription and amplification process, which is about 3.4-fold faster than conventional bulk RT-LAMP. Ultrafast quantification of SARS-CoV-2 is accomplished in 15 min without virus pre-lysis and RNA extraction. The sensitivity can accurately quantify SARS-CoV-2 down to 0.5 copy/μL. Furthermore, the integrated system has an excellent specificity, reproducibility and storage stability, which can be also used to test SARS-CoV-2 on various cold chain fruits. The developed ultrafast and simple hydrogel RT-LAMP will be an enormous potential for surveillance of virus or other hazardous microbes in environmental, agricultural and food industry.

Noninvasive Prenatal Testing of Methylmalonic Acidemia cblC Type Using the cSMART Assay for MMACHC Gene Mutations.

Noninvasive prenatal testing (NIPT) for monogenic disorders has been developed in recent years; however, there are still significant technical and analytical challenges for clinical use. The clinical feasibility of NIPT for methylmalonic acidemia cblC type (cblC type MMA) was investigated using our circulating single-molecule amplification and re-sequencing technology (cSMART). Trios molecular diagnosis was performed in 29 cblC type MMA-affected children and their parents by traditional Sanger sequencing. In the second pregnancy, invasive prenatal diagnosis (IPD) of the pathogenic MMACHC gene was used to determine fetal genotypes, and NIPT was performed using a novel MMACHC gene–specific cSMART assay. Maternal–fetal genotypes were deduced based on the mutation ratio in maternal plasma DNA. Concordance of fetal genotypes between IPD and NIPT, and the sensitivity and specificity of NIPT were determined. After removing two cases with a low P value or reads, the concordance ratio for NIPT and IPD was 100.00% (27/27), and the sensitivity and specificity were 100.00% (54.07–100.00%) and 100.00% (83.89–100.00%), respectively. This study demonstrates that NIPT using the cSMART assay for cblC type MMA was accurate in detecting fetal genotypes. cSMART has a potential clinical application as a prenatal diagnosis and screening tool for carrier and low-risk genotypes of cblC type MMA and other monogenic diseases.

Hydrogel-compartmentalized heterogeneous amplification for viral digital genotyping

Accurate viral genotyping is significant for exploring clinical manifestations and treatment options. The classical nucleic acid amplification within homogeneous system merely reflects the average characteristics of all molecules in samples, which prevents the simultaneous analysis for sample with viruses of different genotypes. Herein, we propose hydrogel-compartmentalized nucleic acid amplification and introduce restriction enzyme to establish a competitive amplification reaction. This system localizes high molecular-weight viral nucleic acids and allows single-molecule heterogeneous amplification in response to various endonuclease digestion. With this strategy, we could provide accurate information for digital genotyping of Hepatitis B virus. Considering its well-performance and easy-to-use, the hydrogel-compartmentalized heterogeneous amplification system should be a promising tool for digital genotyping, and will be of great help to other genetic analysis.

Tracking the evolution of untreated high-intermediate/high-risk diffuse large B-cell lymphoma by circulating tumour DNA.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogenous malignancy, early identification of patients for relapse remains challenging. The potential to non-invasively monitor tumour evolutionary dynamics of DLBCL needs to be further established. In the present study, 17 tumour biopsy and 38 plasma samples from 38 patients with high-intermediate/high-risk DLBCL were evaluated at baseline. Longitudinal blood samples were also collected during therapy. Circulating tumour DNA (ctDNA) was analysed using targeted sequencing based on a gene panel via a recently developed methodology, circulating single-molecule amplification and re-sequencing technology (cSMART). We found that the most frequently mutated genes were tumour protein p53 (TP53; 42·1%), histone-lysine N-methyltransferase 2D (KMT2D; 28·9%), caspase recruitment domain family member 11 (CARD11; 21·1%), cAMP response element-binding protein binding protein (CREBBP; 15·8%), β2 -microglobulin (B2M; 15·8%), and tumour necrosis factor alpha-induced protein 3 (TNFAIP3; 15·8%). The mutation profiles between ctDNA and matched tumour tissue showed good concordance; however, more mutation sites were detected in ctDNA samples. Either TP53 or B2M mutations before treatment predicted poor prognosis. Analysis of dynamic blood samples confirmed the utility of ctDNA for the real-time assessment of treatment response and revealed that the increases in ctDNA levels and changes in KMT2D mutation status could be useful predictors of disease progression. Our present results suggest that ctDNA is a promising method for the detection of mutation spectrum and serves as a biomarker for disease monitoring and predicting clinical recurrence.

The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients.

Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Identification of Somatic Mutations in Papanicolaou Smear DNA and Plasma Circulating Cell-Free DNA for Detection of Endometrial and Epithelial Ovarian Cancers: A Pilot Study.

ObjectivesThe aim of this study was to identify tumor-derived DNA from Papanicolaou (Pap) smear and plasma specimens collected from patients with endometrial cancer or atypical hyperplasia (EC/AH) or epithelial ovarian cancer (OC).MethodsTumor tissues, peripheral blood, and Pap smear samples were collected from patients with EC/AH and patients with epithelial OC. Somatic mutations of tumor specimens in EC/AH and OC were examined by whole-exome sequencing using a 127-driver gene panel from The Cancer Genome Atlas (TCGA). A nine-gene EC/AH panel and an eight-gene OC panel were established based on the identified significantly mutated genes in the EC/AH and OC tumor specimens. Circulating single-molecule amplification and resequencing technology (cSMART) was applied to evaluate somatic mutations in Pap smear DNA and plasma circulating cell-free DNA (ccfDNA) using the EC/AH and OC gene panels.ResultsIn EC/AH group, there existed 22 tumors and 14 of the 22 tumors contributed hot spot mutations for the EC/AH nine-gene panel. In the Pap smear subgroup, all 21 Pap smears tested positive. Nine out of 11 (81.8%) identified the same gene mutations with their matched tumors and the remaining 10 Pap smears all tested positive. In the plasma subgroup, 10 out of 26 (38.5%) plasmas tested positive. One out of 13 (7.7%) identified the same gene mutation with its matched tumor and 5 out of the remaining 13 plasmas (38.5%) tested positive. In OC group, there existed 17 tumors and 16 of the 17 tumors contributed hot spot mutations for the OC eight-gene panel. In the Pap smear subgroup, all 11 Pap smears tested positive. Five out of 10 (50.0%) identified the same gene mutations with their matched tumors and the remaining one Pap smear also tested positive. In the plasma subgroup, all 22 plasmas tested positive. Ten out of 14 (71.4%) identified the same gene mutation with their matched tumors and the remaining 4 plasmas all tested positive.ConclusionsTumor-derived DNA can be detected in Pap smears and plasmas from patients with EC/AH or epithelial OC. Using a small gene-panel, early detection of EC/AH and OC might be promising. However, the value of plasma ccfDNA for EC/AH requires further investigation.

Liquid drop of DNA libraries reveals total genome information

Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

Genetic profiling of primary and secondary tumors from patients with lung adenocarcinoma and bone metastases reveals targeted therapy options

BackgroundPatients newly diagnosed with lung adenocarcinoma with bone metastases (LABM) have poor survival rates after treatment with conventional therapies. To improve outcomes, we retrospectively investigated whether the application of a more comprehensive genetic test of tumor biopsies samples from LABM patients could provide the basis for treatment with more effective tyrosine kinase inhibitors (TKIs) regimens.MethodsFine needle biopsies were taken from the primary tumor (PT) and a secondary bone metastasis (BM) of 17 LABM patients before treatment. Simple genetic profiles for selecting therapies were initially obtained using an ARMS-PCR test for EGFR and ALK fusion mutations. More detailed genetic profiles of somatic exon SNVs and CNVs in 457 cancer-related genes were retrospectively derived using capture single molecule amplification and resequencing technology (capSMART).ResultsARMS-PCR identified 14 EGFR positive, 3 EGFR negative and 1 ALK fusion positive patient. A therapy regimen incorporating TKIs Gefitinib and Crizotinib was offered to the EGFR and ALK fusion positive patients, respectively. With the exception of two patients, molecular profiling of matching PT and BM biopsies identified a highly shared somatic variant fingerprint, although the BMs exhibited additional genomic instability. In six of 13 EGFR positive patients and in all three EGFR negative patients, examination of the genetic profiles identified additional clinically significant mutations that are known or experimental drug targets for treatment of lung cancer.ConclusionOur findings firstly suggest that treatment regimens based on comprehensive genetic assessment of newly diagnosed LABM patients should target both the PT and secondary BMs, including rogue clones with potential to form new BMs. Second, the additional information gained should allow clinicians to design and implement more personalized treatment regimens and potentially improve outcomes for LABM patients.

Circulating Tumor DNA as a Prognostic Biomarker in Localized Non-small Cell Lung Cancer.

BackgroundRoutine clinical surveillance involves serial radiographic imaging following radical surgery in localized non-small cell lung cancer (NSCLC). However, such surveillance can detect only macroscopic disease recurrence and is frequently inconclusive. We investigated if detection of ctDNA before and after resection of NSCLC identifies the patients with risk of relapse, and furthermore, informs about response to management.MethodsWe recruited a total of 77 NSCLC patients. A high-throughput 127 target-gene capture technology and a high-sensitivity circulating single-molecule amplification and resequencing technology (cSMART) assay were used to detect the somatic mutations in the tumor tissues as well as the plasma of NSCLC patients before and after surgery to monitor for minimal residual disease (MRD). Kaplan-Meier and Cox regression analysis were performed to evaluate the relapse-free survival (RFS) and overall survival (OS) of patients with predictor variables.ResultsPatients with a higher stage (III/IV) and preoperative ctDNA-positive status demonstrated a significant 2.8-3.4-fold risk and 3.8-4.0-fold risk for recurrence and death, respectively. Preoperative ctDNA-positive patients associated with a lower RFS (HR = 3.812, p = 0.0005) and OS (HR = 5.004, p = 0.0009). Postoperative ctDNA-positive patients also associated with a lower RFS (HR = 3.076, p = 0.0015) and OS (HR = 3.195, p = 0.0053). Disease recurrence occurred among 63.3% (19/30) of postoperative ctDNA-positive patients. Most of these patients 89.5% (17/19) had detectable ctDNA within 2 weeks after surgery and was identified in advance of radiographic findings by a median of 12.6 months.ConclusionAdvanced stage and preoperative ctDNA-positive are strong predictors of RFS and OS in localized NSCLC patients undergoing complete resection. Postoperative detection of ctDNA increases chance to detect early relapse, thus can fulfill an important role in stratifying patients for immediate further treatment with adjuvant and neoadjuvant therapy.

Noninvasive prenatal diagnosis for pregnancies at risk for β-thalassaemia: a retrospective study.

To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for β-thalassaemia using circulating single molecule amplification and re-sequencing technology (cSMART). Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping. Chinese prenatal diagnostic centres specialising in thalassaemia testing. Chinese carrier couples at high genetic risk for β-thalassaemia. Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell-free DNA. Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD. Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06-100.00%) and 97.26% (95% CI 90.45-99.67%), respectively. This study demonstrates that our cSMART-based NIPD assay for β-thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples. A new noninvasive test for pregnancies at risk for β-thalassaemia.

1475P - Impact of plasma EGFR mutation fractions on response to first generation tyrosine-kinase inhibitor in treatment of naïve non-small cell lung cancer patients

BackgroundAlthough liquid biopsy has been reported in detecting actionable mutations, and is becoming standard of care in non-small cell lung cancer (NSCLC) under certain circumstances, the relationship between plasma mutant allele fractions (MAFs) and treatment responsiveness remains unclear. MethodsThis study enrolled 134 patients with advanced NSCLC. Tumor tissue specimens were collected after surgery, and peripheral blood samples were collected at baseline (before surgery) and during treatments. Amplification-refractory mutation system (ARMS) was performed for EGFR mutation screening in all tumor tissues. Circulating single-molecule amplification and resequencing technology (cSMART) was performed in all cfDNA samples using a panel of nine genes including EGFR. EGFR status were compared between tumor tissue and matched cfDNA samples. Moreover, the association between MAFs of EGFR mutations in cfDNA and the responsiveness to tyrosine-kinase inhibitor (TKI) treatment was analyzed. ResultsEGFR mutations were detected in 56 tumor samples (56/134, ∼41.8%) and 67 matched baseline plasma cfDNA samples (67/134, 50.0%), the concordance rate between tumor and cfDNA samples is 82.8%. Specifically, EGFR exon19 non-frameshift deletions were detected in 24 tumor tissue and 23 matched cfDNA samples (concordance rate 97.8%), EGFR p.L858R mutations were detected in 24 tumor tissue and 22 matched cfDNA samples (concordance rate 97.0%). The overall response rate (ORR) and disease control rate (DCR) for EGFR mutation positive patients (TKI sensitive mutations only) were 47.6% and 73.8%, respectively. More importantly, for TKI-treated patients with original plasma EGFR MAFs higher than 0.1% and lower than 0.1%, the ORRs were 58.6% and 23.1% (17/29 versus 3/13 patients, P=0.047), respectively. ConclusionsIn this study we confirmed that liquid biopsy based screening is reliable for actionable somatic mutation detection. The plasma mutant allele fraction of EGFR driver mutation tend to associate with the responsiveness to TKI targeted treatment. Larger cohorts are needed to clarify the impact of plasma MAFs in predicting treatment responsiveness. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Multimode MicroRNA Sensing via Multiple Enzyme-Free Signal Amplification and Cation-Exchange Reaction.

The detection of biomarkers requires not only high sensitivity but also different signal reading methods depending on the actual situation. Herein, the luminescent properties of CdTe quantum dots (QDs) were exploited, where CdTe QDs were used as shared signal molecules. Combining multiple types of nucleic acid and chemical signal amplification techniques, and various signal detection techniques, a magnetic nanoparticle (NP) and filter-assisted separation multimode sensing strategy has been developed. In this work, miRNA-141 was selected as a representative target, which can trigger the catalyzed hairpin assembly and hybrid chain reaction enzyme-free nucleic acid signal amplification that generates long double-stranded DNA. Subsequently, the chemical amplification of silver NPs (Ag NPs) that release a large amount of Ag+ was introduced into the system. Finally, the cation-exchange reaction between CdTe QDs and Ag+ was utilized to quench the fluorescence (FL) of the CdTe QDs, releasing free Cd2+. The visual/FL/chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS)/inductively coupled plasma mass spectrometry (ICP-MS) method could then be performed for the analysis of miRNA. After investigating its experimental performance, it has been found that 10 fM can be differentiated from the blank solution with the naked eye. In addition, FL/CVG-AFS/ICP-MS methods all displayed good analytical capability for target detection, and the limits of detection (LODs) are as low as fM, which show high target sequence selectivity. This platform was applied to investigate miRNA-141 expression in various cancer cells, which can accurately detect in the range of 100-100 000 MDA-MB-231 cells (breast cancer cell lines), with an LOD of 15 cells. Therefore, the multimode sensing strategy based on a single signal molecule and multiple signal amplification strategies is an applicable and versatile detection method of biomarkers; it can even achieve point-of-care testing, improving the accuracy and efficiency of medical diagnosis.

Clinical implications of monitoring circulating tumor DNA in early- and late-stage non-small cell lung cancer patients.

e20607 Background: Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and the most common types of NSCLC are squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The development of noninvasive methods to monitor circulating tumor DNA (ctDNA) continues to be a major challenge in NSCLC. Methods: We investigated if detection of ctDNA after resection of NSCLC identifies the patients with risk of relapse, and furthermore, informs about response to management.In this cohort study, high-throughput 168 target-gene capture technology and high-sensitivity circulating single molecule amplification and re-sequencing technology (cSMART) were used to detect the somatic mutations in tissues and plasma of patients with NSCLC, respectively. Moreover, ctDNA somatic mutations were used to monitor changes in minimal residual disease during a follow-up period. Results: A total of 169 patients with lung squamous cell carcinoma and adenocarcinoma were included. Detectable levels of ctDNA were present in 60.7% of patients with stage I and 68.8% of patients with late-stage. In patients not treated with adjuvant chemotherapy, ctDNA was detected preoperatively in 46 of 81 (56.8%) patients, 14 (30.4%) of whom had recurred at follow-up of 44 months; recurrence occurred in only 2 (5.7 %) of 35 patients with negative ctDNA. Serial ctDNA status changed from positive to negative during the initial phase of post operation in four patients. Then, ctDNA became positive again after 2 weeks to 3 months, all the four patients with relapse during the follow-up of 44 months. Conclusions: Detection of ctDNA supplies evidence of residual disease and identifies patients at risk of relapse. These observations have implications for the intervention of lung squamous cell carcinoma and adenocarcinoma patients.

NIPT - POTENTIAL TOOL FOR PGT FOLLOW UP

Objective Invasive procedures such as amniocentesis and CVS, in combination with karyotyping or molecular testing, are currently used to confirm PGT results of transferred embryos. However, some couples who have achieved a pregnancy following PGT-M are reluctant to use this confirmatory approach even though the risk of miscarriage and infection associated with fetal cell sampling is low. In this study, we investigate noninvasive prenatal testing (NIPT) as an alternative tool for PGT-M follow up. Materials and Methods NIPT of the cell free fetal DNAwas performed for pregnancies at risk for a single gene disease using circulating single molecule amplification and re-sequencing technology (cSMART) to target the known parental mutations and genome-wide SNPs. Fetal genotypes were called based on the maternal plasma mutation ratios. Results In preliminary studies of naturally conceived pregnancies undergoing testing for autosomal recessive conditions, fetal genotypes predicted by cSMART assay were concordant with invasive results in 4 of 4 cases (100%) of Wilson disease, 18 of 18 cases (100%) of PKU, 73 of 80 cases (91%) of autosomal recessive hearing loss and 98 of 100 cases (98%) of beta thalassemia. There was a strong association of low fetal DNA fraction and poor quality plasma samples with discordant NIPT results. In a further study, we performed NIPT follow up for a complicated PGT-M case for Fanconi Anemia with HLA matching. The case involved a couple who were both carriers of an identical FANCG deletion mutation. Following embryo testing by mutation analysis and linked STR analysis, 2 HLA matched and disease free (normal and carrier) embryos were transferred, resulting in a twin pregnancy. At 15 weeks gestation, cSMART analysis of the pregnancy plasma determined fetal DNA fractions of 14.2% and 6.6% for twin 1 and 2, respectively. The maternal plasma FANCG mutation ratio was measured at 46.2% (50% minus half the fetal fraction of twin 2), which was consistent with the presence of a carrier fetus (twin 1) and a normal fetus (twin 2). Additional retrospective studies of the WGA products from the transferred embryos using single molecule sequencing also confirmed the FANCG genotypes of the transferred embryos and a HLA match to the sick sibling. Conclusions In proof of concept studies for autosomal recessive conditions, we demonstrate that NIPT can be successfully used to reliably and accurately determine fetal genotypes. NIPT has clinical utility as a safe alternative to invasive testing for confirmation of PGT-M cases that result in an ongoing pregnancy.

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.