ABSTRACT
Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.
ABSTRACT
Most studies of ultrasensitive diagnosis of biomolecules from liquid specimens are limited by problems during sample preparation steps, including enrichment and isolation of biomolecules. Here we report a novel platform combining bis(sulfosuccinimidyl)suberate (BS3) and helix-shaped microchannels (BSH) to change the sample preparation paradigm. This BSH system is composed of BS3 for pathogen enrichment and nucleic acid isolation by electrostatic and covalent interaction, and helix-shaped microchannels to minimize sample loss and remove bubbles in large liquid specimens without pH change. The system detected Mycobacterium tuberculosis following enrichment and isolation of 10 mL of liquefied sputum from 11 patients with tuberculosis. Moreover, the system identified KRAS mutations following cell-free DNA isolation of blood plasma from 10 patients with colorectal cancer. This system allows ultrasensitive diagnosis in various disease applications with large volumes of liquid samples.
Subject(s)
Liquid Biopsy/methods , Succinimides/metabolism , HumansABSTRACT
Recent advances in nucleic acid based testing using bio-optical sensor approaches have been introduced but most are based on hybridization between the optical sensor and the bio-molecule and not on an amplification mechanism. Direct nucleic acid amplification on an optical sensor has several technical limitations, such as the sensitivity of the temperature sensor, instrument complexity, and high background signal. We here describe a novel nucleic acid amplification method based on a whispering gallery mode active resonator and discuss its potential molecular diagnostic application. By implanting nanoclusters as active compounds, this active resonator operates without tapered fiber coupling and emits a strong photoluminescence signal with low background in the wavelength of low absorption in an aqueous environment that is typical of biosensors. Our method also offers an extremely low detection threshold down to a single copy within 10 min due to the strong light-matter interaction in a nano-gap structure. We envision that this active resonator provides a high refractive index contrast for tight mode confinement with simple alignment as well as the possibility of reducing the device size so that a point-of-care system with low-cost, high-sensitivity and simplicity.
ABSTRACT
We evaluated the diagnostic performance of a simple and label-free pathogen enrichment method using homobifunctional imidoesters (HI) and a microfluidic system, called the SLIM assay, followed by real-time PCR from cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV)-uninfected patients with suspected tuberculous meningitis (TBM). Patients with suspected TBM were prospectively enrolled in a tertiary hospital in an intermediate tuberculosis (TB)-burden country during a 30-month period. TBM was classified according to the uniform case definition. Definite and probable TBM were regarded as the reference standards for TBM, and possible TBM and not-TBM as the reference standards for not-TBM. Of 72 HIV-uninfected patients with suspected TBM, 10 were diagnosed with definite (n = 2) and probable (n = 8) TBM by the uniform case definition. The sensitivity of the SLIM assay was 100% (95% confidence interval [CI], 69 to 100%) compared with definite or probable TBM, and it was superior to those of mycobacterial culture (20% [95% CI, 3 to 56%]) and the Xpert MTB/RIF assay (0% [95% CI, 0 to 31%]). Of 21 possible TBM and 41 not-TBM patients by the uniform case definition, 5 possible TBM and no not-TBM patients gave positive results in the SLIM assay. The specificity of the SLIM assay was 92% (95% CI, 82 to 97%; 5/62). We demonstrated that the SLIM assay had a very high sensitivity and specificity with small samples of 10 cases of definite or probable TBM. Further studies are needed to confirm this finding and to compare the SLIM assay with mycobacterial culture, Xpert MTB/RIF, and Xpert MTB/RIF Ultra assays in a larger prospective cohort of patients with suspected TBM, including both HIV-infected and HIV-uninfected cases.
Subject(s)
Microfluidics/methods , Tuberculosis, Meningeal/diagnosis , Adult , Aged , DNA, Bacterial/genetics , Female , HIV Infections , Humans , Imidoesters , Male , Middle Aged , Molecular Diagnostic Techniques , Mycobacterium tuberculosis , Prospective Studies , Sensitivity and Specificity , Tertiary Care Centers , Tuberculosis, Meningeal/cerebrospinal fluidABSTRACT
Infectious diseases, especially pathogenic infections, are a growing threat to public health worldwide. Since pathogenic bacteria usually exist in complex matrices at very low concentrations, the development of technology for rapid, convenient, and biocompatible sample enrichment is essential for sensitive diagnostics. In this study, a cucurbit[6]uril (CB) supermolecular decorated amine-functionalized diatom (DA) composite was fabricated to support efficient sample enrichment and in situ nucleic acid preparation from enriched pathogens and cells. CB was introduced to enhance the rate and effectiveness of pathogen absorption using the CB-DA composite. This novel CB-DA composite achieved a capture efficiency of approximately 90% at an Escherichia coli concentration of 106 CFU/mL within 3 min. Real-time PCR analyses of DNA samples recovered using the CB-DA enrichment system showed a four-fold increase in the early amplification signal strength, and this effective method for capturing nucleic acid might be useful for preparing samples for diagnostic systems.
Subject(s)
Biocompatible Materials , Nanocomposites , Specimen Handling , Biocompatible Materials/chemistry , Cell Line, Tumor , Diatoms/chemistry , Humans , Macrocyclic Compounds/chemistry , Microbiological Techniques , Nanocomposites/chemistry , Nanocomposites/ultrastructure , Nucleic Acids/chemistry , Nucleic Acids/isolation & purification , Specimen Handling/methodsABSTRACT
Nucleic acid-based diagnostics are widely used for clinical applications due to their powerful recognition of biomolecule properties. Isolation and purification of nucleic acids such as DNA and RNA in the diagnostic system have been severely hampered in point-of-care testing because of low recovery yields, degradation of nucleic acids due to the use of chaotropic detergent and high temperature, and the requirement of large instruments such as centrifuges and thermal controllers. Here, we report a novel large instrument- and detergent-free assay via binary nanomaterial for ultrasensitive nucleic acid isolation and detection from cells (eukaryotic and prokaryotic). This binary nanomaterial couples a zinc oxide nanomultigonal shuttle (ZnO NMS) for cell membrane rupture without detergent and temperature control and diatomaceous earth with dimethyl suberimidate complex (DDS) for the capture and isolation of nucleic acids (NA) from cells. The ZnO NMS was synthesized to a size of 500 nm to permit efficient cell lysis at room temperature within 2 min using the biological, chemical, and physical properties of the nanomaterial. By combining the ZnO NMS with the DDS and proteinase K, the nucleic acid extraction could be completed in 15 min with high quantity and quality. For bacterial cells, DNA isolation with the binary nanomaterial yielded 100 times more DNA, than a commercial spin column based reference kit, as determined by the NanoDrop spectrophotometer. We believe that this binary nanomaterial will be a useful tool for rapid and sensitive nucleic acid isolation and detection without large instruments and detergent in the field of molecular diagnostics.
Subject(s)
Nanostructures/chemistry , Nucleic Acids/analysis , Polymerase Chain Reaction/methods , Bacteria/genetics , DNA, Bacterial/analysis , DNA, Bacterial/isolation & purification , Detergents/chemistry , Dimethyl Suberimidate/chemistry , Endopeptidase K/metabolism , HCT116 Cells , Humans , Nucleic Acids/isolation & purification , Particle Size , Point-of-Care Testing , Temperature , Zinc Oxide/chemistryABSTRACT
BACKGROUND: Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the most common tick-borne illnesses in South Korea. Early differentiation of SFTS from scrub typhus in emergency departments is essential but difficult because of their overlapping epidemiology, shared risk factors, and similar clinical manifestations. METHODS: We compared the diagnostic performance of one-step isothermal nucleic acid amplification with bio-optical sensor detection (iNAD) under isothermal conditions, which is rapid (20-30 min), with that of real-time PCR, in patients with a confirmed tick-borne illness. Fifteen patients with confirmed SFTS who provided a total of 15 initial blood samples and 5 follow-up blood samples, and 21 patients with confirmed scrub typhus, were evaluated. RESULTS: The clinical sensitivity of iNAD (100%; 95% CI, 83-100) for SFTS was significantly higher than that of real-time PCR (75%; 95% CI, 51-91; P = 0.047), while its clinical specificity (86%; 95% CI, 65-97) was similar to that of real-time PCR (95%; 95% CI, 77-99; P = 0.61). The clinical sensitivity of iNAD for scrub typhus (100%; 95% CI, 81-100) was significantly higher than that of real-time PCR for scrub typhus (67%; 95% CI, 43-85; P = 0.009), while its clinical specificity (90%; 95% CI, 67-98) was similar to that of real-time PCR (95%; 95% CI, 73-100; P > 0.99). CONCLUSIONS: iNAD is a valuable, rapid method of detecting SFTS virus and Orientia tsutsugamushi with high clinical sensitivity and specificity.
Subject(s)
Nucleic Acid Amplification Techniques/methods , Phlebotomus Fever/diagnosis , Scrub Typhus/diagnosis , Tick-Borne Diseases/diagnosis , Aged , Aged, 80 and over , Humans , Middle Aged , Optics and Photonics/instrumentation , Optics and Photonics/methods , Real-Time Polymerase Chain Reaction , Republic of Korea , Retrospective Studies , Sensitivity and SpecificityABSTRACT
Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip. Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10â¯ml urine with Salmonella and 10 times more sensitive in 10â¯ml urine with Brucella than that of real-time PCR without the enrichment process. Furthermore, the combination system of the enrichment platform and an RPA sensor that based on an isothermal DNA amplification method with rapidity and sensitivity for detection can detect a pathogen at down to 50â¯CFU in 10â¯ml urine for Salmonella and 102â¯CFU in 10â¯ml urine for Brucella within 60â¯min. This system will be useful as it has the potential for better diagnosis of pathogens by increasing the capture efficiency of the pathogen in large samples, subsequently enhancing the detection limit of pathogenic DNA.
Subject(s)
Communicable Diseases/diagnosis , DNA, Bacterial/genetics , Microfluidic Analytical Techniques , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Communicable Diseases/geneticsABSTRACT
Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9-mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54â¯aM) and SFTS (0.63â¯aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20â¯min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.
ABSTRACT
The isolation of nucleic acids in the lab on a chip is crucial to achieve the maximal effectiveness of point-of-care testing for detection in clinical applications. Here, we report on the use of a simple and versatile single-channel microfluidic platform that combines dimethyl pimelimidate (DMP) for nucleic acids (both RNA and DNA) extraction without electricity using a thin-film system. The system is based on the adaption of DMP into nonchaotropic-based nucleic acids and the capture of reagents into a low-cost thin-film platform for use as a microfluidic total analysis system, which can be utilized for sample processing in clinical diagnostics. Moreover, we assessed the use of the DMP system for the extraction of nucleic acids from various samples, including mammalian cells, bacterial cells, and viruses from human disease, and we also confirmed that the quality and quantity of the nucleic acids extracted were sufficient to allow for the robust detection of biomarkers and/or pathogens in downstream analysis. Furthermore, this DMP system does not require any instruments and electricity, and has improved time efficiency, portability, and affordability. Thus, we believe that the DMP system may change the paradigm of sample processing in clinical diagnostics.
ABSTRACT
Wound management remains a critical healthcare issue due to the rising incidence of chronic diseases leading to persistent wounds. Traditional dressings have their limitations, such as potential for further damage during changing and suboptimal healing conditions. Recently, hydrogel-based dressings have gained attention due to their biocompatibility, biodegradability, and ability to fill wounds. Particularly, polysaccharide-based hydrogels have shown potential in various medical applications. This study focuses on the development of a novel hydrofilm wound dressing produced from a blend of chia seed mucilage (CSM) and polyvinyl alcohol (PVA), termed CSMP. While the individual properties of CSM and PVA are well-documented, their combined potential in wound management is largely unexplored. CSMP, coupled with sorbitol and glycerin, and cross-linked using ultraviolet light, results in a flexible, adhesive, and biocompatible hydrofilm demonstrating superior water absorption, moisturizing, and antibacterial properties. This hydrofilm promotes epithelial cell migration, enhanced collagen production, and outperforms existing commercial dressings in animal tests. The innovative CSMP hydrofilm offers a promising, cost-effective approach for improved wound care, bridging existing gaps in dressing performance and preparation simplicity. Future research can unlock further applications of such polysaccharide-based hydrofilm dressings.
Subject(s)
Anti-Bacterial Agents , Wound Healing , Animals , Bandages , Cell Movement , Glycerol/pharmacology , Hydrogels/pharmacologyABSTRACT
BACKGROUND: Brain-derived exosomes circulate in the bloodstream and other bodily fluids, serving as potential indicators of neurological disease progression. These exosomes present a promising avenue for the early and precise diagnosis of neurodegenerative conditions. Notably, miRNAs found in plasma extracellular vesicles (EVs) offer distinct diagnostic benefits due to their stability, abundance, and resistance to breakdown. RESULTS: In this study, we introduce a method using transferrin conjugated magnetic nanoparticles (TMNs) to isolate these exosomes from the plasma of patients with neurological disorders. This TMNs technique is both quick (<35 min) and cost-effective, requiring no high-priced ingredients or elaborate equipment for EV extraction. Our method successfully isolated EVs from 33 human plasma samples, including those from patients with Parkinson's disease (PD), Multiple Sclerosis (MS), and Dementia. Using quantitative polymerase chain reaction (PCR) analysis, we evaluated the potential of 8 exosomal miRNA profiles as biomarker candidates. Six exosomal miRNA biomarkers (miR-195-5p, miR-495-3p, miR-23b-3P, miR-30c-2-3p, miR-323a-3p, and miR-27a-3p) were consistently linked with all stages of PD. SIGNIFICANCE: The TMNs method provides a practical, cost-efficient way to isolate EVs from biological samples, paving the way for non-invasive neurological diagnoses. Furthermore, the identified miRNA biomarkers in these exosomes may emerge as innovative tools for precise diagnosis in neurological disorders including PD.
Subject(s)
Exosomes , Magnetite Nanoparticles , MicroRNAs , Parkinson Disease , Transferrin , Humans , Parkinson Disease/diagnosis , Parkinson Disease/blood , Exosomes/chemistry , MicroRNAs/blood , Magnetite Nanoparticles/chemistry , Transferrin/chemistry , Brain/metabolism , Biomarkers/blood , Male , FemaleABSTRACT
Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.
Subject(s)
Nanostructures , Nucleic Acids , Humans , Microfluidics , Silicon Dioxide , Oligonucleotide Array Sequence Analysis/methods , Nucleic Acid Amplification TechniquesABSTRACT
Changes in specific circulating RNA (circRNA) expressions can serve as diagnostic noninvasive biomarkers for prostate cancer (PCa). However, there are still unmet needs, such as unclear types and roles of circRNAs, PCa detection in benign prostatic hyperplasia (BPH) by unstandardized methods, and limitations of sample volume capacity and low circRNA concentrations. This study reports a simple and rapid circRNA enrichment and isolation technique named "HAZIS-CirR" for the analysis of urinary circRNAs. The method utilizes homobifunctional hydrazides with amine-modified zeolite and polyvinylidene fluoride (PVDF) syringe filtration for combining electrostatic and covalent coupling and size-based filtration, and it offers instrument-free isolation of circRNAs in 20 min without volume limitation, thermoregulation, and lysis. HAZIS-CirR has high capture efficiency (82.03%-92.38%) and a 10-fold more sensitive detection limit (20 fM) than before enrichment (200 fM). The clinical utility of HAZIS-CirR is confirmed by analyzing circulating mRNAs and circulating miRNAs in 89 urine samples. Furthermore, three miRNA panels that differentiate PCa from BPH and control, PCa from control, and BPH from control, respectively, are established by comparing miRNA levels. HAZIS-CirR will be used as an optimal and established method for the enrichment and isolation of circRNAs as diagnostic, prognostic, and predictive biomarkers in human cancers.
ABSTRACT
Fungi cause various forms of invasive fungal disease (IFD), and fungal sensitization can contribute to the development of asthma, asthma severity, and other hypersensitivity diseases, such as atopic dermatitis (AD). In this study, we introduce a facile and controllable approach, using homobifunctional imidoester-modified zinc nano-spindle (HINS), for attenuating hyphae growth of fungi and reducing the hypersensitivity response complications in fungi-infected mice. To extend the study of the specificity and immune mechanisms, we used HINS-cultured Aspergillus extract (HI-AsE) and common agar-cultured Aspergillus extract (Con-AsE) as the refined mouse models. HINS composites within the safe concentration range inhibited the hyphae growth of fungi but also reduce the number of fungal pathogens. Through the evaluation of lung and skin tissues from the mice, asthma pathogenesis (lung) and the hypersensitivity response (skin) to invasive aspergillosis were least severe in HI-AsE-infected mice. Therefore, HINS composites attenuate asthma and the hypersensitivity response to invasive aspergillosis.
ABSTRACT
BACKGROUND: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples. METHODS: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers. RESULTS: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases. CONCLUSION: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.
ABSTRACT
Mycobacterium tuberculosis (MTB) is a communicable disease and still remains a threat to common health. Thus, early diagnosis and treatment are required to prevent the spread of infection. Despite the recent advances in molecular diagnostic systems, the commonly used MTB diagnostic tools are laboratory-based assays, such as mycobacterial culture, MTB PCR, and Xpert MTB/RIF. To address this limitation, point-of-care testing (POCT)-based molecular diagnostic technologies capable of sensitive and accurate detection even in environments with limited sources are needed. In this study, we propose simple tuberculosis (TB) molecular diagnostic assay by combining sample preparation and DNA-detection steps. The sample preparation is performed using a syringe filter with amine-functionalized diatomaceous earth and homobifunctional imidoester. Subsequently, the target DNA is detected by quantitative PCR (polymerase chain reaction). The results can be obtained within 2 h from samples with large volumes, without any additional instruments. The limit of detection of this system is 10 times higher than those of conventional PCR assays. We validated the clinical utility of the proposed method in 88 sputum samples obtained from four hospitals in the Republic of Korea. Overall, the sensitivity of this system was superior to those of other assays. Therefore, the proposed system can be useful for MTB diagnosis in limited-resource settings.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Humans , Tuberculosis, Pulmonary/diagnosis , Pathology, Molecular , Sensitivity and Specificity , Sputum/microbiology , Molecular Diagnostic Techniques/methodsABSTRACT
Surface-enhanced Raman scattering (SERS) has evolved into a robust analytical technique capable of detecting a variety of biomolecules despite challenges in securing a reliable Raman signal. Conventional SERS-based nucleic acid detection relies on hybridization assays, but reproducibility and signal strength issues have hindered research on directly amplifying nucleic acids on SERS surfaces. This study introduces a deep learning assisted ZnO-Au-SERS-based direct amplification (ZADA) system for rapid, sensitive molecular diagnostics. The system employs a SERS substrate fabricated by depositing gold on uniformly grown ZnO nanorods. These nanorods create hot spots for the amplification of the target nucleic acids directly on the SERS surface, eliminating the need for postamplification hybridization and Raman reporters. The limit of detection of the ZADA system was superior to those of the conventional amplification methods. Clinical validation of the ZADA system with coronavirus disease 2019 (COVID-19) samples from human patients yielded a sensitivity and specificity of 92.31% and 81.25%, respectively. The integration of a deep learning program further enhanced sensitivity and specificity to 100% and reduced SERS analysis time, showcasing the potential of the ZADA system for rapid, label-free disease diagnosis via direct nucleic acid amplification and detection within 20 min.
Subject(s)
COVID-19 , Deep Learning , Nucleic Acids , Zinc Oxide , Humans , Spectrum Analysis, Raman , Pathology, Molecular , Reproducibility of Results , COVID-19 TestingABSTRACT
Detection of oncogene mutations has significance for early diagnosis, customized treatment, treatment progression, and drug resistance monitoring. Here, we introduce a rapid, sensitive, and specific mutation detection assay based on the hot-spot-specific probe (HSSP), with improved clinical utility compared to conventional technologies. We designed HSSP to recognize KRAS mutations in the DNA of colorectal cancer tissues (HSSP-G12D (GGTâGAT) and HSSP-G13D (GGCâGAC)) by integration with real-time PCR. During the PCR analysis, HSSP attaches to the target mutation sequence for interference with the amplification. Then, we determine the mutation detection efficiency by calculating the difference in the cycle threshold (Ct) values between HSSP-G12D and HSSP-G13D. The limit of detection to detect KRAS mutations (G12D and G13D) was 5-10% of the mutant allele in wild-type populations. This is superior to the conventional methods (≥30% mutant allele). In addition, this technology takes a short time (less than 1.5 h), and the cost of one sample is as low as USD 2. We verified clinical utility using 69 tissue samples from colorectal cancer patients. The clinical sensitivity and specificity of the HSSP assay were higher (84% for G12D and 92% for G13D) compared to the direct sequencing assay (80%). Therefore, HSSP, in combination with real-time PCR, provides a rapid, highly sensitive, specific, and low-cost assay for detecting cancer-related mutations. Compared to the gold standard methods such as NGS, this technique shows the possibility of the field application of rapid mutation detection and may be useful in a variety of applications, such as customized treatment and cancer monitoring.
Subject(s)
Colorectal Neoplasms , ras Proteins , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , Humans , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Real-Time Polymerase Chain Reaction , ras Proteins/genetics , ras Proteins/therapeutic useABSTRACT
BACKGROUND: It is difficult to diagnose tuberculosis (TB), particularly sputum-scarce pulmonary TB and extrapulmonary TB, using conventional diagnostic tests. Since these cases require additional invasive procedures to obtain appropriate specimens, new non-invasive diagnostic tests are needed. Plasma cell-free DNA (cfDNA) detection has gained interest as a novel diagnostic test for TB as it is convenient and less invasive. Therefore, we investigated the performance of enriched cfDNA for diagnosing pulmonary TB and extrapulmonary TB. METHODS: All patients suspected to have TB, who consented to the use of blood for detecting cfDNA, were prospectively enrolled from January 2019 to June 2020. We categorised the patients as confirmed, probable, possible TB, and not-TB. We compared the performance of cfDNA with those of conventional diagnostic tests. RESULTS: Among the 96 patients enrolled, 40 (41.7%) had TB, including 34 with confirmed TB and six probable TB, and 41 (42.7%) did not have TB. Acid-fast bacilli microscopy, Xpert MTB/RIF, and mycobacterial culture results were positive in 12 (31.6%), 22 (61.1%), and 25 (65.8%) patients, respectively. The sensitivity and specificity of cfDNA were 80.0% and 78.1%, respectively. While the sensitivity and specificity of cfDNA were similar to those of interferon-gamma releasing assay (IGRA) (sensitivity 80.6% and specificity 71.4%), the combined sensitivity and specificity of the two assays were 94.4% and 64.3%, respectively, which can be used to rule out TB. CONCLUSIONS: Plasma cfDNA assay seems to be a useful adjunct to the current tests for diagnosing TB, especially when used in combination with IGRA for ruling out TB.AbbreviationsTBtuberculosiscfDNAcell-free DNAPCRpolymerase chain reactionAFBacid-fast bacilliIGRAinterferon-gamma releasing assayCTcomputed tomographyHIVhuman immunodeficiency virus.