scholarly journals A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus

Sensors ◽  
2020 ◽  
Vol 20 (13) ◽  
pp. 3728
Author(s):  
Ching-Chou Wu ◽  
Hao-Yu Yen ◽  
Lu-Ting Lai ◽  
Guey-Chuen Perng ◽  
Cheng-Rei Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Electrochemical Impedance ◽  
Denv Infection ◽  
Viral Disease ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Great Promise ◽  
Label Free ◽  
Hybridization Efficiency

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

scholarly journals A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 479
Author(s):  
Soumi Sukla ◽  
Prasenjit Mondal ◽  
Subhajit Biswas ◽  
Surajit Ghosh
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Magnetic Beads ◽  
Molecular Beacon ◽  
Detection System ◽  
Denv Infection ◽  
Molecular Beacons ◽  
Nucleic Acid Amplification ◽  
Complementary Sequence ◽  
Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

scholarly journals Salivary Detection of Dengue Virus NS1 Protein with a Label-Free Immunosensor for Early Dengue Diagnosis

Sensors ◽  
2018 ◽  
Vol 18 (8) ◽  
pp. 2641 ◽  
Author(s):  
Daniel Wasik ◽  
Ashok Mulchandani ◽  
Marylynn Yates
Keyword(s):  
Early Diagnosis ◽  
Dengue Virus ◽  
Dengue Infection ◽  
Denv Infection ◽  
Human Saliva ◽  
Diagnostic Methods ◽  
Label Free ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Blood Draw

Dengue virus (DENV) is a highly pathogenic, arthropod-borne virus transmitted between people by Aedes mosquitoes. Despite efforts to prevent global spread, the potential for DENV epidemics is increasing world-wide. Annually, 3.6 billion people are at risk of infection. With no licensed vaccine, early diagnosis of dengue infection is critical for clinical management and patient survival. Detection of DENV non-structural protein 1 (NS1) is a clinically accepted biomarker for the early detection of DENV infection. Unfortunately, virtually all of the laboratory and commercial DENV NS1 diagnostic methods require a blood draw for sample analysis, limiting point-of-care diagnostics and decreases patient willingness. Alternatively, NS1 in human saliva has been identified for the potential early diagnosis of DENV infection. The collection of saliva is simple, non-invasive, painless, and inexpensive, even by minimally trained personnel. In this study, we present a label-free chemiresistive immunosensor for the detection of the DENV NS1 protein utilizing a network of single-walled carbon nanotubes functionalized with anti-dengue NS1 monoclonal antibodies. NS1 was successfully detected in adulterated artificial human saliva over the range of clinically relevant concentrations with high sensitivity and selectivity. It has potential application in clinical diagnosis and the ease of collection allows for self-testing, even within the home.

Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

2011 ◽  
Author(s):  
Shinnosuke Inoue ◽  
Woon-Hong Yeo ◽  
Jong-Hoon Kim ◽  
Jae-Hyun Chung ◽  
Kyong-Hoon Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Staphylococcus Epidermidis ◽  
Genomic Dna ◽  
Bacterial Culture ◽  
Low Cost ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

scholarly journals Gene Expression Signatures in AML-12 Hepatocyte Cells upon Dengue virus Infection and Acetaminophen Treatment

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1284
Author(s):  
Jorge G. G. Ferreira ◽  
Sandra G. Gava ◽  
Eneida S. Oliveira ◽  
Izabella C. A. Batista ◽  
Gabriel da R. Fernandes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dengue Virus ◽  
Denv Infection ◽  
Viral Disease ◽  
Biological Processes ◽  
Dengue Virus Infection ◽  
Liver Disorders ◽  
Two Factors ◽  
Hepatocyte Cells

Dengue is an acute viral disease caused by Dengue virus (DENV) and is considered to be the most common arbovirus worldwide. The clinical characteristics of dengue may vary from asymptomatic to severe complications and severe organ impairment, particularly affecting the liver. Dengue treatment is palliative with acetaminophen (APAP), usually known as Paracetamol, being the most used drug aiming to relieve the mild symptoms of dengue. APAP is a safe and effective drug but, like dengue, can trigger the development of liver disorders. Given this scenario, it is necessary to investigate the effects of combining these two factors on hepatocyte homeostasis. Therefore, this study aimed to evaluate the molecular changes in hepatocytes resulting from the association between DENV infection and treatment with sub-toxic APAP concentrations. Using an in vitro experimental model of DENV-2 infected hepatocytes (AML-12 cells) treated with APAP, we evaluated the influence of the virus and drug association on the transcriptome of these hepatocytes by RNA sequencing (RNAseq). The virus–drug association was able to induce changes in the gene expression profile of AML-12 cells and here we highlight and explore these changes and its putative influence on biological processes for cellular homeostasis.

scholarly journals Evaluation of Malaria Diagnostic Methods as a Key for Successful Control and Elimination Programs

2020 ◽  
Vol 5 (2) ◽  
pp. 102
Author(s):  
Afoma Mbanefo ◽  
Nirbhay Kumar
Keyword(s):  
Nucleic Acid ◽  
Diagnostic Tests ◽  
False Negative ◽  
Protein Detection ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
World Health ◽  
Resource Limited ◽  
Successful Control ◽  
Polymerase Chain

Malaria is one of the leading causes of death worldwide. According to the World Health Organization’s (WHO’s) world malaria report for 2018, there were 228 million cases and 405,000 deaths worldwide. This paper reviews and highlights the importance of accurate, sensitive and affordable diagnostic methods in the fight against malaria. The PubMed online database was used to search for publications that examined the different diagnostic tests for malaria. Currently used diagnostic methods include microscopy, rapid diagnostic tests (RDT), and polymerase chain reaction (PCR). Upcoming methods were identified as loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), isothermal thermophilic helicase-dependent amplification (tHDA), saliva-based test for nucleic-acid amplification, saliva-based test for Plasmodium protein detection, urine malaria test (UMT), and transdermal hemozoin detection. RDT, despite its increasing false negative, is still the most feasible diagnostic test because it is easy to use, fast, and does not need expensive equipment. Noninvasive tests that do not require a blood sample, but use saliva or urine, are some of the recent tests under development that have the potential to aid malaria control and elimination. Emerging resistance to anti-malaria drugs and to insecticides used against vectors continues to thwart progress in controlling malaria. Therefore, future innovation will be required to enable the application of more sensitive and affordable methods in resource-limited settings.

scholarly journals Sialic Acid Expression in the MosquitoAedes aegyptiand Its Possible Role in Dengue Virus-Vector Interactions

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Jorge Cime-Castillo ◽  
Philippe Delannoy ◽  
Guillermo Mendoza-Hernández ◽  
Verónica Monroy-Martínez ◽  
Anne Harduin-Lepers ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Aedes Aegypti ◽  
Dengue Virus ◽  
Mammalian Cells ◽  
Vector Competence ◽  
Denv Infection ◽  
Viral Disease ◽  
Salivary Gland Extract ◽  
Denv Serotypes ◽  
Mosquito Saliva

Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquitoAedes aegyptithat has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) inAedes aegyptitissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA forAedes aegyptiCMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells.AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related toα-2,6-sialyltransferase were detected in theAedes aegyptigenome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

scholarly journals Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 412
Author(s):  
Gajanan T. Mehetre ◽  
Vincent Vineeth Leo ◽  
Garima Singh ◽  
Antonina Sorokan ◽  
Igor Maksimov ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Viral Disease ◽  
Plant Viruses ◽  
Diagnostic Methods ◽  
Viral Diseases ◽  
Portable Devices ◽  
Accurate Identification ◽  
Treatment And Prevention ◽  
Short Period

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

scholarly journals Development of a Portable SPR Sensor for Nucleic Acid Detection

Micromachines ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 526 ◽  
Author(s):  
Yafeng Huang ◽  
Lulu Zhang ◽  
Hao Zhang ◽  
Yichen Li ◽  
Luyao Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Incident Angle ◽  
Incident Light ◽  
High Sensitivity ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Nucleic Acid Detection ◽  
Scanning System ◽  
Label Free ◽  
Spr Sensor

Nucleic acid detection is of great significance in clinical diagnosis, environmental monitoring and food safety. Compared with the traditional nucleic acid amplification detection method, surface plasmon resonance (SPR) sensing technology has the advantages of being label-free, having simple operation, and providing real-time detection. However, the angle scanning system in many SPR angle modulation detection applications usually requires a high-resolution stepper motor and complex mechanical structure to adjust the angle. In this paper, a portable multi-angle scanning SPR sensor was designed. The sensor only uses one stepping motor to rotate a belt, and the belt pulls the mechanical linkages of incident light and reflected light to move in opposite directions for achieving the SPR angle scanning mode that keeps the incident angle and reflected angle equal. The sensor has an angle scanning accuracy of 0.002°, response sensitivity of 3.72 × 10−6 RIU (refractive index unit), and an angle scanning range of 30°–74°. The overall size of the system is only 480 mm × 150 mm × 180 mm. The portable SPR sensor was used to detect nucleic acid hybridization on a gold film chip modified with bovine serum albumin (BSA). The result revealed that the sensor had high sensitivity and fast response, and could successfully accomplish the hybridization detection of target DNA solution of 0.01 μmol/mL.

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