A Single Electrochemical Biosensor Designed to Detect Any Virus.

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.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

PURPOSE: In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2. METHODS: Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed. RESULTS: The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively. CONCLUSIONS: The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.

A rapid and high sensitivity RNA detection based on NASBA and G4-ThT fluorescent biosensor.

In recent years, various newly emerged and re-emerged RNA viruses have seriously threatened the global public health. There is a pressing need for rapid and reliable nucleic acid-based assays for detecting viral RNA. Here, we successfully developed a highly sensitive, easy-to-operate G4-ThT-NASBA system to detect viral RNA that no need for labeled primers and probes. Next, we tested the system for detecting the Classical Swine Fever Virus (CSFV), an RNA virus that causes a highly contagious disease in domestic pigs and wild boar and easily causes huge economic losses. Results showed that the system, integrated the G4-ThT fluorescent biosensor and NASBA (Nuclear acid sequence-based amplification),is capable to detect as little as 2 copies/µL of viral RNA without interfering by other swine viral RNA. Moreover, we were able to detect CSFV RNA within 2 h in serum samples taken from the field in a real-time mode. These findings indicate that the G4-ThT-NASBA system is a rapid, high sensitivity and easy-to-operate technique for RNA detection. The method also has the real-time detection capability which may be easily integrated in a highly automated system such as microfluidic chips.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

The mechanism and improvements to the isothermal amplification of nucleic acids, at a glance.

A comparative review of the most common isothermal methods is provided. In the last two decades, the challenge of using isothermal amplification systems as an alternate to the most extensive and long-standing nucleic acids-amplifying method-the polymerase chain reaction-has arisen. The main advantage of isothermal amplification is no requirement for expensive laboratory equipment for thermal cycling. Considerable efforts have been made to improve the current techniques of nucleic acid amplification and the development of new approaches based on the main drawbacks of each method. The most important and challenging goal was to achieve a low-cost, straightforward system that is rapid, specific, accurate, and sensitive.

Isothermal SARS-CoV-2 Diagnostics: Tools for Enabling Distributed Pandemic Testing as a Means of Supporting Safe Reopenings.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

[Application of NASBA and RPA in detection of pathogenic bacteria].

Nucleic acid sequence-based amplification and recombinase polymerase amplification are the recently developed thermostatic amplification techniques based on PCR. This paper briefly summarizes the principle of reaction, design principle of primer and probe, advantage of these two techniques (simple, accurate, highly sensitive and rapid) and introduces the application of the techniques in the detection of pathogenic bacteria.

Pooled analysis of nuclear acid sequence-based amplification for rapid diagnosis of Mycoplasma pneumoniae infection.

BACKGROUND: Mycoplasma pneumoniae (M pneumoniae) is a common human etiology of respiratory infections. Nuclear acid sequence-based amplification (NASBA) shows good value for the detection of M pneumoniae that surpasses PCR. However, the optimal detection technology still remains to be identified. The purpose of this meta-analysis was to systematically evaluate the overall accuracy of NASBA for diagnosing M pneumoniae infections. METHODS: The databases PubMed, Cochrane Library, Google Scholar, CNKI, Wang Fang, and Baidu Scholar were comprehensively searched from their initiation date to December 2017 for NASBA in the diagnosis of M pneumoniae infection. Meta-DiSc 1.4 statistical software was used to evaluate the sensitivity (SEN), specificity (SPE), negative likelihood ratio (-LR), positive likelihood ratio (+LR), diagnostic odds ratio (DOR), and summary receiver operating characteristic (SROC). RevMan 5.2 statistical software was used for quality evaluation of the included articles. Publication bias was evaluated by funnel plot. RESULTS: Six articles with high quality, including 10 studies, were finally included in this meta-analysis. The combined statistics results for the diagnosis of M pneumoniae infection by NASBA were 0.77 (SEN, 95% CI: 0.71 to 0.82); 0.98 (SPE, 95% CI: 0.98 to 0.99); 0.22 (-LR, 95% CI: 0.13 to 0.39); 50.38 (+ LR, 95% CI: 21.85 to 116.17); 292.72 (DOR, 95% CI: 95.02 to 901.75); and 0.9875 (the area under the curve of SROC). CONCLUSION: Nuclear acid sequence-based amplification is a reliable technique to diagnose M pneumoniae infection. However, whether it can replace PCR and serology need to be further studied.

Development of a real-time nucleic acid sequence-based amplification assay for the rapid detection of Salmonella spp. from food.

Salmonella spp. is one of the most common foodborne infectious pathogen. This study aimed to develop a real-time nucleic acid sequence-based amplification (NASBA) assay for detecting Salmonella in foods. Primers and a molecular beacon targeting the Salmonella-specific xcd gene were designed for mRNA transcription, and 48 Salmonella and 18 non-Salmonella strains were examined. The assay showed a high specificity and low detection limit for Salmonella (7 × 10-1 CFU/mL) after 12 h of pre-enrichment. Importantly, it could detect viable cells. Additionally, the efficacy of the NASBA assay was examined in the presence of pork background microbiota; it could detect Salmonella cells at 9.5 × 103 CFU/mL. Lastly, it was successfully used to detect Salmonella in pork, beef, and milk, and its detection limit was as low as 10 CFU/25 g (mL). The real-time NASBA assay developed in this study may be useful for rapid, specific, and sensitive detection of Salmonella in food of animal origin.

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as "red tides", which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 105 cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 106 cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification.

Solid-phase oligonucleotide amplification is of interest because of possible applications to next-generation sequencing, multiplexed microarray-based detection, and cell-free synthetic biology. Its efficiency is, however, less than that of traditional liquid-phase amplification involving unconstrained primers and enzymes, and understanding how to optimize the solid-phase amplification process remains challenging. Here, we demonstrate the concept of solid-phase nucleic acid sequence-based amplification (SP-NASBA) and use it to study the effect of tethering density on amplification efficiency. SP-NASBA involves two enzymes, avian myeloblastosis virus reverse transcriptase (AMV-RT) and RNase H, to convert tethered forward and reverse primers into tethered double-stranded DNA (ds-DNA) bridges from which RNA- amplicons can be generated by a third enzyme, T7 RNA polymerase. We create microgels on silicon surfaces using electron-beam patterning of thin-film blends of hydroxyl-terminated and biotin-terminated poly(ethylene glycol) (PEG-OH, PEG-B). The tethering density is linearly related to the PEG-B concentration, and biotinylated primers and molecular beacon detection probes are tethered to streptavidin-activated microgels. While SP-NASBA is very efficient at low tethering densities, the efficiency decreases dramatically with increasing tethering density due to three effects: (a) a reduced hybridization efficiency of tethered molecular beacon detection probes; (b) a decrease in T7 RNA polymerase efficiency;

A highly specific Escherichia coli qPCR and its comparison with existing methods for environmental waters.

The presence of Escherichia coli in environmental waters is considered as evidence of faecal contamination and is therefore commonly used as an indicator in both water quality and food safety analysis. The long period of time between sample collection and obtaining results from existing culture based methods means that contamination events may already impact public health by the time they are detected. The adoption of molecular based methods for E. coli could significantly reduce the time to detection. A new quantitative real-time PCR (qPCR) assay was developed to detect the ybbW gene sequence, which was found to be 100% exclusive and inclusive (specific and sensitive) for E. coli and directly compared for its ability to quantify E. coli in environmental waters against colony counts, quantitative real-time NASBA (qNASBA) targeting clpB and qPCR targeting uidA. Of the 87 E. coli strains tested, 100% were found to be ybbW positive, 94.2% were culture positive, 100% were clpB positive and 98.9% were uidA positive. The qPCR assays had a linear range of quantification over several orders of magnitude, and had high amplification efficiencies when using single isolates as a template. This compared favourably with qNASBA which showed poor linearity and amplification efficiency. When the assays were applied to environmental water samples, qNASBA was unable to reliably quantify E. coli while both qPCR assays were capable of predicting E. coli concentrations in environmental waters. This study highlights the inability of qNASBA targeting mRNA to quantify E. coli in environmental waters, and presents the first E. coli qPCR assay with 100% target exclusivity. The application of a highly exclusive and inclusive qPCR assay has the potential to allow water quality managers to reliably and rapidly detect and quantify E. coli and therefore take appropriate measures to reduce the risk to public health posed by faecal contamination.

Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification.

Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card.

In Silico Probe-Based Detection of Citrus Viruses in NGS Data.

The conservation of plant biosecurity relies on the rapid identification of pathogenic organisms, including viruses. With next-generation sequencing (NGS), it is possible to identify multiple viruses within a metagenomic sample. In this study, we explored the use of electronic probes (e-probes) for the simultaneous detection of 11 recognized citrus viruses. E-probes were designed and screened against raw sequencing data to minimize the bioinformatic processing time required. The e-probes were able to accurately detect their cognate viruses in simulated datasets, without any false negatives or positives. The efficiency of the e-probe-based approach was validated with NGS datasets generated from different RNA preparations: double-stranded RNA (dsRNA) from 'Mexican' lime infected with different Citrus tristeza virus (CTV) genotypes, dsRNA from field samples, and small RNA and total RNA from grapefruit infected with the CTV T3 genotype. A set of probes was made available that is able to accurately detect CTV in sequence data regardless of the input dataset or the genotype that plants are infected with.

Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA).

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/µL within 5h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n=103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture.

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

BACKGROUND: The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. CONCLUSION: These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity.

BACKGROUND: Quantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared. METHODS: To quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed. RESULTS: Intra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (< 1 gametocyte per µL). Logistic models, including log-transformed gametocytaemia estimated by QT-NASBA, explained variability in mosquito feeding experiment results as well as log-transformed gametocytaemia by qRT-PCR (marginal R2 0.28 and 0.22, respectively). Densities determined by both methods strongly correlated with mosquito infection rates [Spearman's rank correlation coefficient, 0.59 for qRT-PCR and 0.64 for QT-NASBA (P < 0.001 for both)]. Gametocyte densities estimated by qRT-PCR were higher than levels estimated by QT-NASBA or light microscopy at high densities (>100 gametocyte per µL). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze-thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain. CONCLUSIONS: The experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.

Evaluación de técnicas de amplificación de ácido nucleico (NAT) para detección del virus de la inmunodeficiencia humana, hepatitis C y hepatitis B en bancos de sangre / Assessment on nucleic acid amplification techniques (nat) for human immunodeficiency, hepatitis C and hepatitis B virus screening in blood banks

CONTEXTO CLÍNICO: Las transfusiones de sangre y hemoderivados constituyen una terapéutica frecuente en la actualidad. Sin embargo, si no se toman las medidas necesarias, quienes reciben las donaciones pueden contraer ciertas enfermedades. Disminuir los riesgos de la transmisión de enfermedades infecciosas es un requisito indispensable para los servicios de medicina transfusional.Para tender a la eliminación de este riesgo, se pueden implementar tres tipos de estrategias: a) tratar los componentes con métodos para inactivación de patógenos, con la finalidad de eliminar agentes infecciosos, b) implementar políticas para la captación del donante no relacionado, habitual y responsable y c) aplicar métodos para detección de infecciones transmisibles con una mayor sensibilidad. En relación a esto último, la detección de marcadores serológicos para infecciones transmisibles por transfusión es muy eficiente y ha reducido de manera importante el impacto de esta complicación transfusional en las últimas décadas.1, 2 Sin embargo, existe un período de ventana, en el cual un donante infectado y potencialmente infectante, no presenta niveles dosables de marcadores serológicos; por este motivo se ha postulado el uso de técnicas que permitan la detección en este período de ventana. Un ejemplo de ellos es la técnica NAT (por sus siglas en inglés: nucleic acid amplification Technology), la cual es una prueba de biología molecular que se ha postulado a nivel mundial desde 1999, para la detección del material genético de los virus de la inmunodeficiencia humana (VIH), hepatitis C (VHC) y hepatitis B (VHB) con el fin de disminuir el período de ventana de no-detección, en las muestras provenientes de donantes infectados. TECNOLOGÍA: NAT se realiza mediante la realización de los siguientes pasos básicos: la preparación de la muestra, la amplificación de secuencias de ácido nucleico, y la detección de productos de amplificación (amplicons). OBJETIVO: Evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura de la técnica de amplificación de ácido nucleico en forma rutinaria para la detección del virus de la inmunodeficiencia humana, el virus de la hepatitis C y el de la hepatitis B en muestras de sangre de donantes. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas (incluyendo Medline, Cochrane y CRD), en buscadores genéricos de Internet, agencias de evaluación de tecnologías sanitarias y financiadores de salud utilizando la siguiente estrategia: (Nucleic Acid Amplification Techniques[Mesh:NoExp] OR Amplification Tech*[tiab] OR Nucleic-Acid Amplificat*[tiab] OR DNA Amplificat*[tiab] OR RNA Amplificat*[tiab] OR NAT[tiab]) AND (Blood Donors[Mesh] OR Blood Don*[tiab] OR Blood Banks[Mesh] OR Blood Bank*[tiab]). Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias y económicas, guías de práctica clínica y políticas de cobertura de otros sistemas de salud cuando estaban disponibles. RESULTADOS: Para el siguiente informe se incluyeron dos estudios observacionales, una revisión no sistemática, cuatro GPC, una ETS, seis normas y dos evaluaciones económicas. CONCLUSIONES: Si bien existe escasa evidencia de moderada calidad metodológica que muestra que la utilización de la técnica de amplificación de ácido nucleico (NAT) es efectiva en la detección de los virus de la inmunodeficiencia humana, hepatitis C y hepatitis B en el período ventana serológico, la magnitud del beneficios de su aplicación en forma rutinaria en muestras de sangre de donantes no ha sido aún aclarada. Si bien el uso de NAT se encuentra muy extendido en el mundo y muchas normativas y guías internacionales lo recomiendan; su implementación no es mandatorio en muchos otros países. La Organización Mundial de la Salud aún no lo recomienda en forma rutinaria para todos los países, sobre todo debido a que se estima que los potenciales beneficios de su aplicación son bajos a expensas de un alto costo. Su uso estaría más justificado en contextos con una mayor prevalencia e incidencia de infecciones transmisibles por la sangre.