ABSTRACT
Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5'-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.
Subject(s)
Biosensing Techniques , Viruses , Animals , Humans , Sensitivity and Specificity , Self-Sustained Sequence Replication/methods , RNA, Viral/genetics , RNA, Viral/analysis , Viruses/genetics , Nucleic Acid Amplification TechniquesABSTRACT
DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.
Subject(s)
Biosensing Techniques , Nucleic Acids , RNA, Ribosomal, 16S , DNA/chemistry , DNA Probes/chemistry , Nanotechnology , Electrochemical Techniques , Nucleic Acid Hybridization , Methylene Blue/chemistryABSTRACT
A highly reproducible electrochemical biosensor, employing a five-stranded four-way junction (5S-4WJ) system through square wave voltammetry, has been successfully validated for the detection of Influenza A virus (InfA). A comprehensive assessment of its linearity, precision, accuracy, and robustness has demonstrated its compliance with FDA standards. Integration with Nucleic Acid-Based Amplification (NASBA) has showcased its selectivity for InfA, enabling the detection of InfA RNA with a standard heater set at 41 °C. This platform offers a straightforward setup well-suited for use at low-resource facilities.
Subject(s)
Biosensing Techniques , Influenza A virus , Influenza A virus/genetics , RNA , Nucleic Acid Amplification TechniquesABSTRACT
A modular, multi-purpose, and cost-effective electrochemical biosensor based on a five-stranded four-way junction (5S-4WJ) system was developed for SARS-CoV-2 (genes S and N) and Influenza A virus (gene M) detection. The 5S-4WJ structure consists of an electrode-immobilized universal stem-loop (USL) strand, two auxiliary DNA strands, and a universal methylene blue redox strand (UMeB). This design allows for the detection of specific nucleic acid sequences using square wave voltammetry (SWV). The sequence-specific auxiliary DNA strands (m and f) ensure selectivity of the biosensor for target recognition utilizing the same USL and UMeB components. An important feature of this biosensor is the ability to reuse the USL-modified electrodes to detect the same or alternative targets in new samples. This is accomplished by a simple procedure involving rinsing the electrodes with water to disrupt the 5S-4WJ structure and subsequent re-hybridization of the USL strand with the appropriate set of strands for a new analysis. The biosensor exhibited minimal loss in signal after rehybridization, demonstrating its potential as a viable multiplex assay for both current and future pathogens, with a low limit of quantification (LOQ) of as low as 17 pM.
Subject(s)
COVID-19 , Influenza, Human , Humans , SARS-CoV-2 , Cost-Benefit Analysis , Influenza, Human/diagnosis , ElectrodesABSTRACT
No disponible
Subject(s)
Humans , Male , Female , Primary Health Care/methods , Primary Health Care/trends , Integrated Advanced Information Management Systems/trends , Delivery of Health Care, Integrated/methods , Delivery of Health Care, Integrated , Primary Health Care/organization & administration , Primary Health CareABSTRACT
In Chile swine trichinosis has presented a progressive decreasing in the last two decades of XX century. T. spiralis pig infection descended from an average of 0,683 per 1000 in 1980-1984 to 0,315 in 1985-1989 and to 0,115 in 1990-1996. In the particular case of Metropolitan Region this decreasing has been more marked: from an average of 0,058 per 1000 in 1990-1994 to 0,003 in 1995-1999. Between the end of june 1999 and middle january 2000 in Metropolitan Region abattoirs T. spiralis was detected in 15 (4,9 percent) out of 306 swine from two pigsties located in El Monte (E.M) and Padre Hurtado (P.H) 45 and 30 km south-west from Santiago. In the same period another four pigs from the same premises were found infected in abattoirs of other regions. During inspection visits it was stated that both pig farms had deficient sanitary conditions. Phototrichinoscopy was positive in three out of five rattus norvegicus collected in E.M. In pigsty PH the examination of diaphragm samples of 25 dogs and 17 cats resulted negative. In the premises originating T. spirali infected swine the Metropolitan Environmetal Health Service Abattoirs Program carries out and epidemiological vigilance consisting in the follow-up of animls destined for slaughtering in order to initiate prophylactic actions oriented to eliminate eventual sources of trichinosis infection for human and rearing pigs