-
Genome Medicine May 2022Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the...
BACKGROUND
Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens.
METHODS
We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21).
RESULTS
The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). The ability to predict resistance was concentration-dependent and successful in 7/10 (70%), 18/25 (72%), and 3/21 (14%) of samples with stool PCR concentration thresholds of > 100 femtogram per microliter (fg/μl), 1 to 100 fg/μl, and < 1 fg/μl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84).
CONCLUSIONS
tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.
Topics: Antitubercular Agents; Child; DNA; Humans; Mycobacterium tuberculosis; Pathology, Molecular; Prospective Studies; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Tuberculosis
PubMed: 35585607
DOI: 10.1186/s13073-022-01054-6 -
Biochemistry. Biokhimiia Dec 2016The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization"... (Review)
Review
The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.
Topics: Antibodies; Antigen-Antibody Complex; DNA; Enzyme-Linked Immunosorbent Assay; Norovirus; Polymerase Chain Reaction; Toxins, Biological
PubMed: 28260495
DOI: 10.1134/S0006297916130113 -
Proceedings of the National Academy of... Dec 2012Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single...
Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10-60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
Topics: Blotting, Western; Cell Extracts; Electrophoresis, Polyacrylamide Gel; HIV Antibodies; Humans; Microfluidics; Proteins; Reproducibility of Results
PubMed: 23223527
DOI: 10.1073/pnas.1207754110 -
Cells Jun 2021Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack...
Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.
Topics: Antineoplastic Agents; Cell Culture Techniques; Cell Proliferation; HeLa Cells; Humans; Imaging, Three-Dimensional; Interferometry; Single-Cell Analysis; Staining and Labeling
PubMed: 34209893
DOI: 10.3390/cells10071635 -
Talanta Dec 2023An analytical method based on triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS) was developed for simultaneous determination of femtogram levels...
An analytical method based on triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS) was developed for simultaneous determination of femtogram levels of Np, Pu, and Pu in environmental samples. By carefully controlling the valence states of Np and Pu in the entire separation procedure using a simple single extraction chromatographic column (TK200), the consistent behavior of Np and Pu was achieved, allowing for the reliable application of Pu as the chemical yield tracer for Np, Pu, and Pu. A high decontamination factor of 3.2 × 10 for the most critical interfering element, uranium, was achieved in the chemical separation step. The interferences of UH and peak tailing of U during the measurement of plutonium isotopes were effectively eliminated by utilizing 7.5 mL/min He-1.1 mL/min CO as reaction gases in the octupole collision/reaction cell and employing sequential quadrupole mode for mass separation in ICP-MS/MS. Specifically, the interference of UH was reduced to 10 and the peak tailing of U to 10, surpassing the performance of measurement method without reaction gases by 3 orders of magnitude. The developed method enables the accurate determination of femtogram levels of Np, Pu, and Pu in the samples with U/Np and U/Pu atom ratios of up to 10 and 10, respectively. The developed method was validated by analyzing standard reference materials and spiked soil samples.
PubMed: 37348353
DOI: 10.1016/j.talanta.2023.124798 -
ACS Sensors Mar 2022Rapid, sensitive, on-site identification of SARS-CoV-2 infections is an important tool in the control and management of COVID-19. We have developed a surface-enhanced...
Rapid, sensitive, on-site identification of SARS-CoV-2 infections is an important tool in the control and management of COVID-19. We have developed a surface-enhanced Raman scattering (SERS) immunoassay for highly sensitive detection of SARS-CoV-2. Single-chain Fv (scFv) recombinant antibody fragments that bind the SARS-CoV-2 spike protein were isolated by biopanning a human scFv library. ScFvs were conjugated to magnetic nanoparticles and SERS nanotags, followed by immunocomplex formation and detection of the SARS-CoV-2 spike protein with a limit of detection of 257 fg/mL in 30 min in viral transport medium. The assay also detected B.1.1.7 ("alpha"), B.1.351 ("beta"), and B.1.617.2 ("delta") spike proteins, while no cross-reactivity was observed with the common human coronavirus HKU1 spike protein. Inactivated whole SARS-CoV-2 virus was detected at 4.1 × 10 genomes/mL, which was 10-100-fold lower than virus loads typical of infectious individuals. The assay exhibited higher sensitivity for SARS-CoV-2 than commercial lateral flow assays, was compatible with viral transport media and saliva, enabled rapid pivoting to detect new virus variants, and facilitated highly sensitive, point-of-care diagnosis of COVID-19 in clinical and public health settings.
Topics: COVID-19; Humans; Point-of-Care Systems; SARS-CoV-2; Single-Chain Antibodies; Spike Glycoprotein, Coronavirus
PubMed: 35271769
DOI: 10.1021/acssensors.1c02664 -
PeerJ 2016High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis...
High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100-1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.
PubMed: 27688978
DOI: 10.7717/peerj.2486 -
Applied Spectroscopy Aug 2022Femtosecond laser-induced breakdown spectroscopy (fs-LIBS) is employed to detect tiny amounts of mass ablated from macroscopic specimens and to measure chemical images...
Femtosecond Single-Pulse and Orthogonal Double-Pulse Laser-Induced Breakdown Spectroscopy (LIBS): Femtogram Mass Detection and Chemical Imaging with Micrometer Spatial Resolution.
Femtosecond laser-induced breakdown spectroscopy (fs-LIBS) is employed to detect tiny amounts of mass ablated from macroscopic specimens and to measure chemical images of microstructured samples with high spatial resolution. Frequency-doubled fs-pulses (length 400 fs, wavelength 520 nm) are tightly focused with a Schwarzschild microscope objective to ablate the sample surface. The optical emission of laser-induced plasma (LIP) is collected by the objective and measured with an echelle spectrometer equipped with an intensified charge-coupled device camera. A second fs-laser pulse (1040 nm) in orthogonal beam arrangement is reheating the LIP. The optimization of the experimental setup and measurement parameters enables us to record single-pulse fs-LIBS spectra of 5 nm thin metal layers with an ablated mass per pulse of 100 femtogram (fg) for Cu and 370 fg for Ag films. The orthogonal double-pulse fs-LIBS enhances the recorded emission line intensities (two to three times) and improves the contrast of chemical images in comparison to single-pulse measurements. The size of ablation craters (diameters as small as 1.5 µm) is not increased by the second laser pulse. The combination of minimally invasive sampling by a tightly focused low-energy fs-pulse and of strong enhancement of plasma emission by an orthogonal high-energy fs-pulse appears promising for future LIBS chemical imaging with high spatial resolution and with high spectrochemical sensitivity.
PubMed: 34494912
DOI: 10.1177/00037028211042398 -
Life (Basel, Switzerland) Oct 2023is the largest genus of medically significant fungi. Although most of its members are commensals, residing harmlessly in human bodies, some are opportunistic and... (Review)
Review
is the largest genus of medically significant fungi. Although most of its members are commensals, residing harmlessly in human bodies, some are opportunistic and dangerously invasive. These have the ability to cause severe nosocomial candidiasis and candidemia that affect the viscera and bloodstream. A prompt diagnosis will lead to a successful treatment modality. The smart solution of biosensing technologies for rapid and precise detection of species has made remarkable progress. The development of point-of-care (POC) biosensor devices involves sensor precision down to pico-/femtogram level, cost-effectiveness, portability, rapidity, and user-friendliness. However, futuristic diagnostics will depend on exploiting technologies such as multiplexing for high-throughput screening, CRISPR, artificial intelligence (AI), neural networks, the Internet of Things (IoT), and cloud computing of medical databases. This review gives an insight into different biosensor technologies designed for the detection of medically significant species, especially and , and their applications in the medical setting.
PubMed: 37895480
DOI: 10.3390/life13102099 -
Analytical Chemistry Mar 2022Microfluidic paper-based analytical devices (μPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A μPAD for automating the...
Microfluidic paper-based analytical devices (μPADs) are promising biosensors that may be used in a variety of bioanalytical applications. A μPAD for automating the competitive enzyme-linked immunosorbent assay (ELISA) of small-sized target detection at the femtogram level using submicroliter samples is reported in this study. The proposed μPAD was integrated with a sucrose valve to automate the sequential delivery of reagents, providing simple control of reagent delivery time and simple operation. The use of a sample solution dropping location at the zones on the device that had been prepared with an antibody-conjugated enzyme before immersion in a running buffer allowed minimization of sample volume to 0.6 μL, while eliminating the possible loss of a target molecule by adsorption on the membrane, thus improving detection sensitivity. Furthermore, the proposed device was successfully applied to the automation of competitive ELISA for the detection of aflatoxin B (AFB), a potent carcinogen that causes substantial health risks to humans worldwide, with a detection limit of 60 femtograms or 0.1 ng/mL. The method developed in this study provides high sensitivity, small sample volume, on-site and equipment-free measurements, low-cost operation, and user-friendliness. This approach could be used to analyze small-sized molecules in the fields of food safety and quality control, environmental monitoring, and clinical diagnostics.
Topics: Aflatoxin B1; Enzyme-Linked Immunosorbent Assay; Humans; Lab-On-A-Chip Devices
PubMed: 35302345
DOI: 10.1021/acs.analchem.1c05401