RESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease, and there are critical interests in detecting multiple biomarkers as a single biomarker detection cannot reflect the exact phase of the disease. Exosomes derived from different types of AML cells contain respective combinations of cluster of differentiation (CD) markers that may be used to guide the molecular typing of AML in the clinic. Here, aiming to build more precise molecular typing of AML, we demonstrate multiplex immuno-PCR (mI-PCR) assay for simultaneous detection of multiple surface CDs on exosomes of AML via capillary electrophoresis with laser-induced fluorescence (CE-LIF). This method comprises of four steps: (1) chemical attachment of reporter DNA sequence to the specific detection antibodies, (2) binding of the detection antibodies to their targets on the exosomes, (3) DNA amplification of the reporter DNA, and (4) capillary electrophoresis analysis of the PCR products. With the method, we first realized simultaneous detection of five target CD molecules (CD9, CD34, c-Kit/CD117, CD123, and FLT-3/CD135) on leukemia cell-derived exosomes with high detection sensitivity. The limit of detection (LOD) and limit of quantification (LOQ) are 2.41 ± 0.04 particles/µL and 8.02 ± 0.16 particles/µL, respectively, for leukemia cell-derived exosomes. This mI-PCR is found sensitive enough to detect picogram (10-12) levels of protein concentrations with high recovery (95%) in spiked serum sample experiments. We thus anticipate that the proposed method is promising in sensitive detection of multitargets to assist in the precise molecular typing of many complex diseases.
Assuntos
Diferenciação Celular/fisiologia , Leucemia , Reação em Cadeia da Polimerase Multiplex/métodos , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Eletroforese Capilar/métodos , Fluorescência , Humanos , LasersRESUMO
The escalating concern surrounding microplastic (MP) pollution necessitates urgent attention and the development of rapid techniques for quantifying extremely low concentrations. Surface-enhanced Raman spectroscopy (SERS) has emerged as a promising method due to its simplicity, high sensitivity, and rapid quantification capabilities. Herein, the efficacy of gold-silver alloy nanoparticles (3DPAu-Ag) substrates for detecting poly(methyl methacrylate) (PMMA) and polystyrene (PS) MPs is investigated. The 3DPAu-Ag SERS substrates are fabricated using the meniscus-confined electrochemical 3D printing (MC-E3DP) process, employing a nozzle of 0.8 mm size with 2.5 V potential at a printing speed of 0.4 mm s-1. The proposed SERS substrates exhibit exceptional sensitivity and are capable of detecting PMMA concentrations as low as 0.2 µg mL-1 and PS concentrations of 1.2 µg mL-1 within the ranges of 1-103 µg mL-1 and 10-104 µg mL-1, respectively. Remarkable enhancement factors (EFs) of up to 3.2 × 104 for PMMA and 9.3 × 103 for PS are achieved, underscoring the substrates' effectiveness. Furthermore, the investigation demonstrates outstanding uniformity and reproducibility of the 3DPAu-Ag substrates, with relative standard deviation (RSD) values of only 4.1 and 6.4%, respectively, across 31 and 5 measurements. Additionally, a minimal 17% decrease in the initial SERS signal value after 5 weeks highlights the substrates' high stability. This not only highlights the superior quality of the substrates but also positions them ahead of previously reported works in the literature. Moreover, this study also comes up with a plausible mechanism for MPs SERS detection facilitated by the 3DPAu-Ag substrates, offering insights into the underlying processes. Overall, 3DPAu-Ag substrates show promise for sensitive, stable MP detection, which is crucial for environmental monitoring.
RESUMO
A multitargeted loop-mediated isothermal amplification (MT-LAMP) assay targeting mpt64 (Rv1980c) and IS6110 was designed to diagnose genitourinary tuberculosis (GUTB) cases. While assessing gel-based, hydroxynaphthol blue (HNB) and SYBR Green I MT-LAMP assays on GUTB specimens (n = 28) in a pilot study, both gel-based/SYBR Green I assays exhibited better sensitivity than HNB LAMP. Since SYBR Green MT-LAMP is easier to perform compared with a gel-based assay, a higher number of GUTB specimens (n = 55) were evaluated by SYBR Green MT-LAMP, wherein 85.5% sensitivity and 94.4% specificity (n = 36) were obtained. Moreover, the sensitivity attained by MT-LAMP was significantly higher (p < 0.05) than with multiplex-PCR (mpt64 + IS6110). After further validating these MT-LAMP data in different epidemiological settings, this assay may be developed as a diagnostic kit.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Tuberculose , Benzotiazóis , Diaminas , Humanos , Técnicas de Diagnóstico Molecular , Projetos Piloto , Quinolinas , Sensibilidade e EspecificidadeRESUMO
This article reports an amide based Chemosensor used for selective detection of divalent Cu+2 and Ni+2 ions via Fluorescence turn off. The selective sensing ability of Chemosensor was investigated in presence of different metal ions Mg2+, Ag+, Fe2+, K+, Cu2+, Ni2+, Hg2+, Pb2+, Mn2+, Pd2+, Cd2+ and Mn3+ as competitive ions. The receptor i. e. Chemosensor formed complexes with metal ions in 1:1 stoichiometric ratio. The detection limit and binding constant calculated as 1.92×10-4 and 1.4×10-4 M and 2.16×103 M-1 and 3.09×103 M-1 for Cu2+ and Ni2+ions respectively. The complexes were characterized by UV/visible, FT-IR, 13C NMR and 1H NMR spectroscopy. Further the structure and Crystallinity were calculated by P-XRD spectral analysis. The crystallinity found to be 65.27 and 67.87% respectively.
Assuntos
Cobre/análise , Sondas Moleculares/química , Níquel/análise , Cátions Bivalentes/análise , Cátions Bivalentes/química , Cobre/química , Fluorescência , Limite de Detecção , Espectroscopia de Ressonância Magnética/métodos , Níquel/química , Piridinas/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Tiazóis/químicaRESUMO
Extracellular vesicles (EVs), the small circulating vesicles released from urine samples of tuberculosis (TB) patients, contain a pool of biomarkers. We recently detected Mycobacterium tuberculosis lipoarabinomannan (LAM) and CFP-10 (Rv3874) biomarkers from the urinary EVs of pulmonary TB (PTB) and extrapulmonary TB (EPTB) patients by immuno-polymerase chain reaction (I-PCR) assay and the results were compared with the analogous enzyme-linked immunosorbent assay (ELISA). The detection limits of both purified LAM and CFP-10 were determined to be 1 fg/mL with I-PCR, which was 106 times lower than ELISA. Detection of LAM and CFP-10 biomarkers in urinary EVs of TB patients by I-PCR showed superiority over ELISA. Notably, LAM I-PCR revealed sensitivities of 74.3 and 67.9% in PTB (n = 74) and EPTB (n = 53) patients, respectively, with specificities of 91.5-92.8% (n = 116). Moreover, the sensitivities attained with LAM I-PCR were significantly higher (P < 0.01) than with CFP-10 I-PCR. After further improving the sensitivity and specificity of the assay, our I-PCR based on LAM detection in urinary EVs may be used as an adjunct test for rapid diagnosis of TB.
Assuntos
Proteínas de Bactérias/análise , Vesículas Extracelulares/química , Lipopolissacarídeos/análise , Tuberculose/patologia , Urinálise , Urina/química , Biomarcadores/urina , Testes Diagnósticos de Rotina/métodos , Humanos , Sensibilidade e Especificidade , Tuberculose/diagnósticoRESUMO
PURPOSE: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. METHODS: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. RESULTS: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. CONCLUSION: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Ouro/química , Fenômenos Magnéticos , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase/métodos , Humanos , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Mycobacterium tuberculosis/imunologia , Tuberculose/diagnósticoRESUMO
Exosomes as nanosized biomarkers hold great potential for the diagnosis of cancer. However, the low concentration of cancer-derived exosomes present in biofluids makes early diagnosis strenuous. Here, we developed a fluorescent biosensing platform, namely a dual signal amplification, for the ultrasensitive detection of leukemia cell-derived exosomes. The protocol consists of three steps: first, leukemia-derived exosomes containing CD63 and nucleolin were captured by anti-CD63 antibody modified magnetic bead conjugates (MB-CD63); then, a DNA primer comprising a nucleolin-recognition aptamer (AS1411) was applied to bind the exosomes which further initiated a rolling circle amplification (RCA) reaction to generate many repeat sequences for hybridization with gold nanoparticle (GNP)-DNA-fluorescent dye (FAM) conjugates (GNP-DNA-FAM); finally, nicking endonuclease (Nb·BbvCI) assisted target recycling was introduced. As a result, FAM was released from GNP-DNA-FAM conjugates, transformed from the quenching state to the emission state and thus fluorescence signals continuously accumulated. With this dual signal amplification platform, as low as 1 × 102 particles per µL exosomes could be detected. Furthermore, we have successfully applied this method for the detection of exosomes in spiked serum samples, indicating a promising tool for clinical application.
Assuntos
Técnicas Biossensoriais , Exossomos/metabolismo , Corantes Fluorescentes/química , Espectrometria de Fluorescência , Tetraspanina 30/metabolismo , Anticorpos/química , Anticorpos/imunologia , DNA/química , DNA/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , Exossomos/química , Ouro/química , Células HL-60 , Humanos , Leucemia/metabolismo , Leucemia/patologia , Magnetismo , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , Tetraspanina 30/imunologiaRESUMO
AIM: Diagnosis of pleural TB poses serious challenges due to paucibacillary nature of specimens and there is an urgent need to devise a reliable diagnostic test. METHODS: We compared GeneXpert Mycobacterium tuberculosis/rifampin assay and the multiplex PCR (M-PCR) targeting mpb64 (Rv1980c) and IS6110 in pleural fluids (n = 78) of pleural TB patients and non-TB controls. RESULTS: The sensitivities of 89.6 and 33.3%, and specificities of 96.7 and 100%, were observed with M-PCR and Xpert assay, respectively. CONCLUSION: M-PCR showed superiority over Xpert assay and may facilitate an efficient diagnosis of pleural TB.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/isolamento & purificação , Derrame Pleural/microbiologia , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Proteínas de Bactérias/genética , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
A novel indirect real-time immuno-polymerase chain reaction (RT-I-PCR) assay, an evolution of I-PCR, was developed for the quantitative detection of Mycobacterium tuberculosis PstS1 (Rv0934) with a wide dynamic range of 10ng/mL to 1pg/mL in body fluids of tuberculosis (TB) patients, which may monitor the dynamics of disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose/diagnóstico , Adolescente , Adulto , Técnicas de Tipagem Bacteriana , Estudos de Casos e Controles , Contagem de Colônia Microbiana , DNA Bacteriano/isolamento & purificação , Estudos de Avaliação como Assunto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Tuberculose/microbiologia , Adulto JovemRESUMO
Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in the development of rapid antigen detection methods. However, the World Health Organization (WHO) has emphasized to continue research for designing new antibody-based detection tests with improved accuracy. Immuno-polymerase chain reaction (I-PCR) combines the simplicity and versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification capacity and sensitivity of PCR thus leading to several-fold increase in sensitivity in comparison to analogous ELISA. In this review, we have described the serodiagnostic potential of I-PCR assays for an early diagnosis of TB based on the detection of potential mycobacterial antigens and circulating antibodies in body fluids of TB patients.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Reação em Cadeia da Polimerase/métodos , Testes Sorológicos/métodos , Tuberculose Pulmonar/diagnóstico , Tuberculose/diagnóstico , Animais , Anticorpos/química , Anticorpos Antibacterianos/análise , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , DNA Bacteriano , Humanos , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologiaRESUMO
A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Fatores Corda/imunologia , Mycobacterium tuberculosis/imunologia , Reação em Cadeia da Polimerase/métodos , Testes Sorológicos/métodos , Tuberculose/diagnóstico , Aciltransferases/sangue , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos , Antígenos de Bactérias/sangue , Proteínas de Bactérias/sangue , Fatores Corda/sangue , Ditiotreitol/química , Ditiotreitol/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina G/química , Maleimidas , Pessoa de Meia-Idade , Tuberculose/sangue , Tuberculose/imunologia , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/imunologia , Adulto JovemRESUMO
Attempts were made to enhance the sensitivity of immuno-PCR assay based on the detection of cocktail of mycobacterial antigen 85B (Rv1886c), ESAT-6 (Rv3875) and cord factor (trehalose 6,6'-dimycolate) in pulmonary and extrapulmonary TB patients. Detection of Ag85B was found to be superior to the detection of cocktail in TB patients.
Assuntos
Antígenos de Bactérias/análise , Fatores Corda/análise , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Antígenos de Bactérias/imunologia , Fatores Corda/imunologia , Imunoensaio/métodos , Tuberculose/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologiaRESUMO
PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Técnicas Bacteriológicas/métodos , Elementos de DNA Transponíveis/genética , DNA Bacteriano/análise , DNA Bacteriano/genética , Diagnóstico Precoce , Feminino , Amplificação de Genes , Humanos , Masculino , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/normas , Sensibilidade e EspecificidadeRESUMO
We developed a novel indirect sandwich immuno-polymerase chain reaction (I-PCR) assay for the detection of mycobacterial antigen 85B (Ag85B, 30kDa, Rv1886c) in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients. The amino-modified reporter DNA was covalently attached with the antidetection antibody through a heterobifunctional cross-linking agent succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. The detection limit of Ag85B by I-PCR was found to be 1 femtogram (fg)/mL, which was 10(6)-fold lower than an analogous enzyme-linked immunosorbent assay (ELISA). The sensitivities of 85% and 77% with I-PCR and 77.6% and 62.5% with ELISA were observed in smear-positive and smear-negative PTB patients, respectively, with high specificity. On the other hand, sensitivities of 84% and 63.7% with I-PCR and 68% and 47.5% with ELISA were observed in confirmed and clinically suspected EPTB cases, respectively, with high specificity.
Assuntos
Aciltransferases/análise , Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Imunoensaio/métodos , Reação em Cadeia da Polimerase/métodos , Tuberculose/diagnóstico , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens. A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets.