Target Amplification Research Articles

HPV Infection and Detection

Human papillomavirus (HPV) can be transmitted by sexual activity and direct skin-to-skin contact. The virus accounts for more than 90% of cervical cancer cases. Target amplification and signal amplification are two detection methods for HPV diagnosis. COBAS 4800 and the Hybrid Capture (HC2) system are two examples of target amplification and signal amplification techniques, respectively. According to the cohort study done in Hong Kong, detection assays have better concordance with samples in the more severe lesion. While COBAS 4800 has greater specificity in targeting high-grade lesions, HC2 has better sensitivity. This review aims to discuss HPV infection, and detection techniques and point out possible improvements.

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Illustrating Heterogeneity in AML By Leveraging Multimodal Analysis of DNA, Surface Proteins, and Intracellular Proteins in Single Cells

Introduction: Recent advancements in precision medicine, although highly promising, present a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multiomics analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights into cellular and genetic composition. Methods: Using a two-step droplet microfluidic technology, the Mission Bio TapestriⓇ platform enables high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a novel workflow was developed to detect protein expression in addition to DNA genotypes in the same single cells. In this approach, cells are labeled with oligonucleotide-conjugated antibodies prior to single-cell encapsulation and DNA target amplification on the Tapestri system. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. The number of protein targets can range from a few targets to over 40, which is beyond the limit for a single flow cytometry run. Results: This workflow has been successfully demonstrated on cell lines as well as clinical samples. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data were able to clearly distinguish CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs from CD34hiCD11blo and CD34loCD11bhi subpopulations that did contain the same pathogenic SNVs and CNVs. Conclusion: We further developed this workflow to include simultaneous detection of intracellular proteins and surface proteins along with targeted sequencing in single cells. The inclusion of intracellular protein detection enables measurement of pivotal proteins in cancer mechanisms. We believe that this novel multiomic technology will facilitate new discoveries inthe complex relationship between genotype and phenotype, enabling a better understanding of disease biology, and subsequently improve the design of diagnostics and therapies.

Development of a PCR assay for the detection of human herpes virus type 7.

A PCR assay has been developed to identify the DNA of the human herpes virus type 7. The search and selection of conserved regions was carried out by comparing the whole genome nucleotide sequences of HHV-7. A fragment duplicated in the HHV-7 genomes was chosen as a target for amplification. The performance of the assay was tested on a synthetic matrix and clinical samples. The developed assay has high sensitivity and specificity and showed good efficiency in detecting HHV-7 DNA in clinical samples.

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management

In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis.

Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants. To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis. Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform. The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy. Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.

Reverse transcriptase-free detection of viral RNA using Hemo Klentaq DNA polymerase

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.

An Ultrasensitive Electrochemical Biosensor Integrated by Nicking Endonuclease-Assisted Primer Exchange Reaction Cascade Amplification and DNA Nanosphere-Mediated Electrochemical Signal-Enhanced System for MicroRNA Detection.

Specific and sensitive microRNAs (miRNAs) detection is essential to early cancer diagnosis. The development of these technologies including functional nuclease-mediated target amplification and DNA nanotechnology possesses tremendous potential for the high-performance detection of miRNAs in the accurate diagnosis of disease. In this study, we have established an ultrasensitive electrochemical biosensor by combining nicking endonuclease-assisted primer exchange reaction (PER) cascade amplification with a DNA nanosphere (DNS)-mediated electrochemical signal-enhanced system for the detection of miRNA-21 (miR-21). The cascade amplification is initiated by a nicking endonuclease that can cleave specific DNA substrates and highly amplify translation of the target to single-stranded DNA fragments (sDNA). Then, the PER cascade is powered by strand-displacing polymerase and generates a large amount of nascent single-stranded connector DNA (cDNA) via sDNA triggering of the dumbbell probe (DP), thus achieving the cascade amplification of the target. Finally, the DNS loaded with plenty of electroactive substances can be captured on the electrode via cDNA for further enhancing the electrochemical signal and highly sensitive detection of miR-21. The proposed electrochemical biosensor exhibits a wide detection range of 1 aM to 0.1 nM and a low detection limit of 0.58 aM. The excellent selectivity allows the biosensor to discriminate miR-21 from other miRNAs, even the one base-mismatched sequence. Moreover, the practicability of the biosensor is investigated by analyzing miR-21 in human serum and cancer cell lysate. Therefore, our proposed nicking endonuclease-assisted PER cascade amplification strategy provides a powerful platform for the early detection of miRNA-related disease and molecular diagnosis.

AML-160 Multimodal Analysis of DNA, Surface Proteins, and Intracellular Proteins in Single Cells

Recent advancements in precision medicine, although highly promising, present a major technical challenge to researchers due to disease heterogeneity. The emergence of single-cell technologies has greatly refined the resolution in which sample diversity can be investigated, enhancing the efficiency of selecting appropriate molecular targets. Additionally, applying multiomics analysis on single cells would further improve the understanding of cell-to-cell heterogeneity by providing unique insights into cellular and genetic composition. Using a two-step droplet microfluidic technology, the Mission Bio Tapestri® platform enables high-throughput targeted DNA sequencing in single cells to obtain single-nucleotide variation (SNV) and copy number variation (CNV) information. By leveraging this technology, a novel workflow was developed to detect protein expression in addition to DNA genotypes in the same single cells. In this approach, cells are labeled with oligonucleotide-conjugated antibodies prior to single-cell encapsulation and DNA target amplification on the Tapestri system. Sequencing libraries are then prepared from both antibody oligonucleotides and the amplified DNA sequences, followed by identification of single-cell DNA genotypes and protein signatures from the sequencing readout. The number of protein targets can range from a few targets to over 40, which is beyond the limit for a single flow cytometry run. This workflow has been successfully demonstrated on cell lines as well as clinical samples. In an acute myeloid leukemia (AML) sample, combined single-cell SNV, CNV, and protein expression data illustrated the heterogeneity within the sample. The data were able to clearly distinguish CD3+ T cells and CD19+ B cells without pathogenic SNVs and CNVs from CD34hiCD11blo and CD34loCD11bhi subpopulations that did contain the same pathogenic SNVs and CNVs. We further developed this workflow to include simultaneous detection of intracellular proteins and surface proteins along with targeted sequencing in single cells. The inclusion of intracellular protein detection enables measurement of pivotal proteins in cancer mechanisms. We believe that this novel multiomic technology will facilitate new discoveries in the complex relationship between genotype and phenotype, enabling a better understanding of disease biology, and subsequently improve the design of diagnostics and therapies.

Phasing of de novo mutations using a scaled-up multiple amplicon long-read sequencing approach.

De novo mutations (DNMs) play an important role in severe genetic disorders that reduce fitness. To better understand their role in disease, it is important to determine the parent-of-origin and timing of mutational events that give rise to these mutations, especially in sex-specific developmental disorders such as male infertility. However, currently available short-read sequencing approaches are not ideally suited for phasing, as this requires long continuous DNA strands that span both the DNM and one or more informative single-nucleotide polymorphisms. To overcome these challenges, we optimized and implemented a multiplexed long-read sequencing approach using Oxford Nanopore technologies MinION platform. We focused on improving target amplification, integrating long-read sequenced data with high-quality short-read sequence data, and developing an anchored phasing computational method. This approach handled the inherent phasing challenges of long-range target amplification and the normal accumulation of sequencing error associated with long-read sequencing. In total, 77 of 109 DNMs (71%) were successfully phased and parent-of-origin identified. The majority of phased DNMs were prezygotic (90%), the accuracy of which is highlighted by an average mutant allele frequency of 49.6% and standard error of 0.84%. This study demonstrates the benefits of employing an integrated short-read and long-read sequencing approach for large-scale DNM phasing.

Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.

Hourglass-mimicking biosensor based on disposable centrifugal tube for bacterial detection in large-volume sample

An hourglass-mimicking biosensor was developed to detect target bacteria in 15 mL centrifugal tube using immune magnetic nanobeads to isolate the target from large-volume sample, gold@platinum nanozymes to label the target for amplification of biological signals, and a microplate reader/colorimetric card for determination of the target. First, a centrifugal tube with an iron ball framework was first coaxially assembled in the center of a Halbach ring magnet. After immune magnetic nanobeads, gold@platinum nanozymes and bacterial sample were mixed by repeated bottom-up of the tube using a stepper motor, nanobead-bacteria-nanozyme complexes were formed. Then, colorless H2O2-TMB was catalyzed by the nanozymes to produce blue TMBox. The color change was finally analyzed using the microplate reader or colorimetric card to determine bacterial concentration. This hourglass-mimicking biosensor could separate ∼95% targets from 10 mL bacteria sample and detect targets from 1.6 × 101 to 1.6 × 106 CFU/mL in 1.0 h with low detection limit of 16 CFU/mL.

Detection of Giardia duodenalis and Toxoplasma gondii in soil and water samples in the Quindío River basin, Colombia

Two zoonotic protozoan pathogens, Giardia duodenalis and Toxoplasma gondii, are important causes of waterborne infections in the Quindío region in Colombia. No previous data exist on how contamination occurs at the source for drinking water consumed by the human population in this region. Our aim was to describe the frequency of G. duodenalis and T. gondii DNA in 11 sampling points during a five-month period in water and adjacent soil at the Quindío River basin (Andean region in the central western part of Colombia). The study employed nested PCR for T. gondii, using the B1 gene as the amplification target, and single-round PCR for G. duodenalis assemblage A and assemblage B, amplifying the gdh gene, followed by DNA sequencing. In 50 soil samples, 28% (14/50) were positive for T. gondii. For G. duodenalis, distribution was in equal parts for assemblage A (8%; 4/50) and assemblage B (8%, 4/50). Genotyping of T. gondii sequences showed two soil samples with type I strain, another two samples of soil with type III strain, but most samples were of unidentified strains. In water samples, T. gondii was detected in 9.1% (5/55), G. duodenalis assemblage A in 34.5% (19/55), and G. duodenalis assemblage B in 12.7% (7/55). T. gondii DNA positivity was associated with lower soil temperature (p = 0.0239). Presence of G. duodenalis and T. gondii was evidenced in soil and water samples in the Quindío River basin, indicating soil as the potential source of contamination for the river that it is destined for human consumption. Monitoring these protozoa in drinking water is necessary to prevent public health risks in human populations.

High-Throughput Cell-Free Screening of Eukaryotic Membrane Proteins in Lipidic Mimetics.

Membrane proteins (MPs) carry out important functions in the metabolism of cells, such as the detection of extracellular activities and the transport of small molecules across the plasma and organelle membranes. Expression of MPs for biochemical, biophysical, and structural analysis is in most cases achieved by overexpression of the desired target in an appropriate host, such as a bacterium. However, overexpression of MPs is usually toxic to the host cells and can lead to aggregation of target protein and to resistance to detergent extraction. An alternative to cell-based MP expression is cell-free (CF), or in vitro, expression. CF expression of MPs has several advantages over cell-based methods, including lack of toxicity issues, no requirement for detergent extraction, and direct incorporation of target proteins in various lipidic mimetics. This article describes a high-throughput method for the expression and purification of eukaryotic membrane proteins used in the author's lab. Basic Protocol 1 describes the selection and cloning of target genes into appropriate vectors for CF expression. Basic Protocol 2 describes the assembly of CF reactions for high-throughput screening. Basic Protocol 3 outlines methods for purification and detection of target proteins. Support Protocols 1-6 describe various accessory procedures: amplification of target, treatment of vectors to prepare them for ligation-independent cloning, and the preparation of S30 extract, T7 RNA polymerase, liposomes, and nanodiscs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Target selection, construct design, and cloning into pET-based expression vectors Support Protocol 1: Amplification of target DNA Support Protocol 2: Preparation of ligation-independent cloning (LIC)-compatible vectors Basic Protocol 2: Assembly of small-scale cell-free reactions for high-throughput screening Support Protocol 3: Preparation of Escherichia coli S30 extract Support Protocol 4: Preparation of T7 RNA polymerase Support Protocol 5: Preparation of liposomes Support Protocol 6: Preparation of nanodiscs Basic Protocol 3: Purification and detection of cell-free reaction products.

Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software—An automatic NGS-STR workflow from DNA to profile for forensic science

The Ion Torrent ™ Genexus ™ Sequencer (Genexus) is a highly integrated instrument that can automate library construction, templating, and sequencing in a single-instrument run. By programing the ForeNGS Analysis Software (FNAS), we bridged the gap between sequencing and genotyping without manual intervention. FNAS can automatically transfer sequencing output files from Genexus, analyze the repeat and flanking regions aligned to the GRCh38 assembly, name the alleles according to the ISFG guidelines, and generate user-friendly interactive profiles. Genexus and FNAS can accomplish the fully automatic DNA-to-Profile workflow in forensics. Based on our experiences, the optimal assay parameters on Genexus were validated as follows: 24 cycles of target amplification for library construction; 40 μL of library and 400 bp of template size for templating; 852 flows of dNTPs by order of Ion samba HID2 for sequencing; and 750,000 reads per sample at minimum for 16 samples multiplexed on a lane. By developmental validations of the Precision ID Globalfiler ™ NGS STR Panel v2, Genexus presented competitive performance at the optimal assay parameters qualified to detect commonly used forensic STR markers. It could produce repeatable and reproducible results, and human profiles could be easily separated from nonhuman profiles. Additionally, Genexus was sensitive enough to detect samples with 100 pg of input DNA, and it was suitable for various types of case samples, especially for low copy number samples and degraded samples. Moreover, minor contributors could be detected between the 4:1 and 1:4 mixtures with an analysis threshold of 50 × . The Genexus workflow is a robust and labor-effective solution enabling forensic scientists to obtain NGS-STR profiles within a single day and with only the need to prepare DNA extracts, then set up Genexus, and finally interpret profiles on FNAS.

Programmable Analysis of MicroRNAs by Thermus thermophilus Argonaute-Assisted Exponential Isothermal Amplification for Multiplex Detection (TEAM).

The simultaneous analysis of the levels of multiple microRNAs (miRNAs) is critical to the early diagnosis of cancer. However, this analysis is challenging because of the low concentrations of miRNAs and their high sequence homology. Here, we report a general and programmable diagnostic strategy for miRNA analysis: Thermus thermophilus Argonaute (TtAgo)-assisted exponential isothermal amplification for multiplex detection (TEAM). This system combines exponential isothermal amplification (EXPAR), for target amplification, with programmable TtAgo cleavage, for the generation of the reporting signal. The TEAM assay achieved attomolar sensitivity with a rapid turnaround time (30-35 min). Because of the single-nucleotide precision of TtAgo, the system demonstrated robust multiplex capability in the simultaneous detection of four miRNA targets and the classification of let-7 family members. The TEAM assay was superior in differentiating colorectal cancer patients from healthy individuals relative to the conventional EXPAR and reverse transcription polymerase chain reaction (RT-PCR) methods. This tunable and scalable approach is a powerful nucleic acid analysis tool that holds promise in scientific and clinical applications.

Evaluation of filter paper to transport oro/nasopharyngeal samples to detect SARS-CoV-2 by RT-qPCR

PurposeTo evaluate filter paper as a means to transport oro/nasopharyngeal samples from laboratories with few resources for SARS-CoV-2 detection by RT-qPCR in a central laboratory that usually performs this technique as routine. MethodsA total of 40 specimens were evaluated in parallel by RT-qPCR carried out after RNA extraction using two different protocols: direct RNA extraction (Protocol A - reference method) and RNA extraction after impregnation in filter paper (Protocol B). ResultsThe RT-qPCR for SARS-CoV-2 using Protocol B presented 97.22% (35/36) of agreement for SARS-CoV-2-positive samples when compared to the reference method (Protocol A), even for specimens with low viral load (increased Ct values). Noteworthy, three clinical specimens which were categorized as inconclusive by Protocol A presented amplification of both N1 and N2 targets using Protocol B, presenting positive results for SARS-CoV-2. ConclusionThe use of filter paper to transport oro/nasopharyngeal clinical samples presented very satisfactory results to detect SARS-CoV-2 by RT-qPCR. In addition, it proved to be a feasible and sensitive approach, being able to generate the detection of SARS-CoV-2 even at low concentrations, without presenting false-negative results.

Metal-organic framework-enabled surface state passivation integrating with single-nuclease-propelled multistage amplification for ultrasensitive lab-on-paper photoelectrochemical biosensing

The surface states and photoinduced corrosion in the photoelectrode can severely reduce the photocurrent signal and stability of photoelectrochemical (PEC) sensor, which imposes a great challenge on the improvement of analytical performance. Herein, metal–organic framework (MOF)-enabled surface passivation strategy is proposed to passivate surface states, suppress photocorrosion and facilitate interfacial charge transport. In the concrete, zeolitic imidazolate framework-8 (ZIF-8), which serves as the passivation layer, is in-situ assembled on the surface of paper-based ZnO nanorods (NRs) array to restrain the charge trapping at the surface states and keep ZnO NRs from photocorrosion, thereby substantially enhancing the photocurrent signal and durability. Moreover, the unique single-nuclease-propelled multistage amplification (SNMA) and triple-helix molecular switch (THMS)-conformation-transition-mediated signal quenching strategies are also designed to further improve the sensitivity. The SNMA process can convert target microRNA-141 into quadruple amount of useful output DNA strands (trigger DNA) in one duplex-specific nuclease (DSN) cleavage cycle assisted by succedent DSN cleavage of DNA duplexes, guaranteeing a higher target amplification efficiency. Subsequently, the THMS structure which anchors double amount of signal enhancers to the photoelectrode surface can be disassembled by the resulted trigger DNA, therefore achieving powerful signal amplification. Profiting from the MOF-enabled surface state passivation and elegantly designed signal amplification strategies, the ultrasensitive detection of microRNA-141 is realized with a liner range from 0.2 fM to 2 nM and detection limit of 72 aM, which provides a promising paradigm for developing high-performance lab-on-paper PEC biosensing platform.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index

IntroductionAntimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. MethodsPatients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. ResultsWe included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. ConclusionsWe can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

High efficiency electrochemiluminescence of 3D porous g-C3N4 with dissolved O2 as co-reactant and its sensing application for ultrasensitive detection of microRNA in tumor cells

In this work, the electrochemiluminescence (ECL) phenomenon of three-dimensional graphitic carbon nitride (3D g-C3N4) was reported. Firstly, the proposed 3D g-C3N4 possessed 3D porous interconnected open-framework which enabled faster charge transport and efficient penetration of co-reactants due to “pore confinement effect”. Then, we found that the dissolved O2 could serve as an excellent co-reactant for cathodic ECL of 3D g-C3N4. And the high specific surface area was beneficial to better adsorbing and gathering of dissolved O2 and reactive oxygen species (ROSs), which made them full contact on the surface or inside of 3D g-C3N4, giving a more sufficient ECL reaction and higher ECL signal. Based on the proposed 3D g-C3N4-O2 ECL system, a sensitive biosensor was constructed for microRNA-21 (miRNA-21) detection with assistance of 3D spherical tracks assisted 3D DNA walking machine, which exhibited superior performance for miRNA-21 with detection limit of 0.22 fM. The proposed 3D g-C3N4-O2 ECL system with high ECL efficiency and the effective target conversion and amplification strategies were beneficial to construct ultra-sensitive ECL sensing platform, which would be better applied to clinical bioanalysis.