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1.
Sci Rep ; 14(1): 12125, 2024 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-38802360

RESUMO

Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25 copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15 copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30 min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.


Assuntos
COVID-19 , Colorimetria , Ouro , Nanopartículas Metálicas , Nasofaringe , RNA Viral , SARS-CoV-2 , Sensibilidade e Especificidade , Colorimetria/métodos , Humanos , COVID-19/diagnóstico , COVID-19/virologia , Nanopartículas Metálicas/química , Ouro/química , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Limite de Detecção , Sondas de Oligonucleotídeos/genética , Teste de Ácido Nucleico para COVID-19/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Teste para COVID-19/métodos
2.
J Trop Med ; 2023: 9326183, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38028028

RESUMO

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per µL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per µL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.

3.
BMC Infect Dis ; 22(1): 847, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371168

RESUMO

Nanobiosensor platforms have emerged as convenient and promising approaches with remarkable efficacy for the diagnosis of infectious diseases. Gold nanoparticles (AuNPs) have been widely used due to numerous advantageous properties such as optical, electrical, physicochemical and great biomolecules binding capabilities. This study aimed to apply AuNP-Probe Conjugate for the detection of Leishmania spp., using colorimetric and amplification methods targeting parasitic ITS2 fragment. The first method was carried out by hybridization of 10µL of DNA with 4 µL of probe and addition of 5 µL of 0.2 N HCl (non-amplification method). Second method was followed by polymerase chain reaction (PCR) amplification using thiolated primer, 5 µL of AuNP and 5 µL of 0.2 N HCl. The appearance of red and purple colors indicated positive and negative results, respectively. The minimum of detection for non-amplification and amplification methods for three strains of Leishmania namely L. major, L. tropica and L. infantum were determined to be 32 fg/µL and 16 fg/µL, respectively. Sensitivity for detection of visceral leishmaniasis (VL) for non-amplification and amplification methods included 96% and 100%, respectively and for cutaneous leishmaniasis (CL) included 98% and 100%, respectively. The results of this investigation revealed that sensitivity of amplification method was the same as RT-qPCR, while that of non-amplification method was lower. However, this method was promising because of no need for any equipment, high specificity, enough sensitivity, low cost and rapidity (less than 30 min) to complete after genomic DNA extraction.


Assuntos
Leishmania infantum , Leishmania major , Leishmania tropica , Leishmaniose Cutânea , Leishmaniose Visceral , Nanopartículas Metálicas , Humanos , Ouro , Leishmania tropica/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Cutânea/diagnóstico , Leishmania major/genética , Reação em Cadeia da Polimerase em Tempo Real , Leishmania infantum/genética
4.
J Cell Physiol ; 237(10): 3752-3767, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35959643

RESUMO

Cancer cells must overcome a variety of external and internal stresses to survive and proliferate. These unfavorable conditions include the accumulation of mutations, nutrient deficiency, oxidative stress, and hypoxia. These stresses can cause aggregation of misfolded proteins inside the endoplasmic reticulum. Under these conditions, the cell undergoes endoplasmic reticulum stress (ER-stress), and consequently initiates the unfolded protein response (UPR). Activation of the UPR triggers transcription factors and regulatory factors, including long noncoding RNAs (lncRNAs), which control the gene expression profile to maintain cellular stability and hemostasis. Recent investigations have shown that cancer cells can ensure their survival under adverse conditions by the UPR affecting the expression of lncRNAs. Therefore, understanding the relationship between lncRNA expression and ER stress could open new avenues, and suggest potential therapies to treat various types of cancer.


Assuntos
Neoplasias , RNA Longo não Codificante , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/genética
5.
Cytokine Growth Factor Rev ; 65: 61-74, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35597701

RESUMO

The assertion that a significant portion of the mammalian genome has not been translated and that non-coding RNA accounts for over half of polyadenylate RNA have received much attention. In recent years, increasing evidence proposes non-coding RNAs (ncRNAs) as new regulators of various cellular processes, including cancer progression and nerve damage. Apoptosis is a type of programmed cell death critical for homeostasis and tissue development. Cancer cells often have inhibited apoptotic pathways. It has recently been demonstrated that up/down-regulation of various lncRNAs in certain types of tumors shapes cancer cells' response to apoptotic stimuli. This review discusses the most recent studies on lncRNAs and apoptosis in healthy and cancer cells. In addition, the role of lncRNAs as novel targets for cancer therapy is reviewed here. Finally, since it has been shown that lncRNA expression is associated with specific types of cancer, the potential for using lncRNAs as biomarkers is also discussed.


Assuntos
Neoplasias , RNA Longo não Codificante , Animais , Apoptose , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêutico , RNA Mensageiro
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