Isothermal Nucleic Acid Amplification Assay Research Articles

Viral diarrheas - newer advances in diagnosis and management.

Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas. Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures. Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease.

An Alternative Rapid Confirmation Method for Identifying Listeria monocytogenes from a Variety of 125 g Food Samples Within Two Days of a PCR Presumptive Positive

Cultural confirmation following detection of a Listeria monocytogenespresumptive positive can take 3–7 days to finalize; this uncertainty is a point of frustration for food producers needing to make time-sensitive disposition decisions. To address the demand for shortened time-to-results, an alternative L. monocytogenes confirmation method consisting of two components, (i) a secondary screen using a different rapid method, and (ii) concurrent cultural isolation followed by next-day colony identification was evaluated. For the study, four food matrices (hot dogs, peanut butter, frozen vegetables, and multicomponent frozen meals) were inoculated with low levels (0.36–1.39 MPN/125 g) of L. monocytogenes per the AOAC guidelines for a matrix study. Analyses were performed on 125 g test portions and started with a PCR primary screen (Bio-Rad iQ-Check Listeria monocytogenes II). Next, all enriched food samples underwent a secondary screen by bioMérieux’s GENE-UP LMO2 Real-Time PCR and VIDAS LMX ELFA along with streaking onto RAPID’L.mono Agar. Presumptive positive L. monocytogenes colonies were identified utilizing a high throughput rapid identification method (Hygiena’s BAX System L. monocytogenes Real-Time PCR assay, Neogen’s ANSR isothermal nucleic acid amplification assay, and Bruker’s MALDI Biotyper). Importantly, this study evaluated multiple commercially available options for the secondary screen (n = 2) and rapid identification (n = 3) to allow for easy adoption by testing laboratories. Overall, there was no statistically significant difference (p ≤ 0.05) between the number of L. monocytogenes-positive 125 g samples obtained by the cultural reference method and the alternative confirmation methods (regardless of which method combinations were evaluated). Additionally, this study supports that, when both the primary and secondary screen methods yield a positive result, the sample could be considered a confirmed positive for L. monocytogenes.

A robust, low-cost instrument for real-time colorimetric isothermal nucleic acid amplification.

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay.

Current HIV antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitored drug dispensing, which are not reliable. We report a proof-of-concept adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP) - an NRTI that functions as a deoxyadenosine triphosphate (dATP) analog and long-term adherence marker for PrEP - by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification assay commonly used for point-of-care diagnostics. We present and validate a model that predicts the amplification probability as a function of dATP and TFV-DP concentrations, nucleotide insertion sites on the RNA template, and RNA template concentration. The model can be used to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay may be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance. Due to its simple instrumentation and short runtime (<1 hour), this assay has the potential for implementation in low-complexity laboratories or point-of-care settings, which may improve access to ART and PrEP adherence monitoring.

Rapid, Cheap, and Effective COVID-19 Diagnostics for Africa.

Background: Although comprehensive public health measures such as mass quarantine have been taken internationally, this has generally been ineffective, leading to a high infection and mortality rate. Despite the fact that the COVID-19 pandemic has been downgraded to epidemic status in many countries, the real number of infections is unknown, particularly in low-income countries. However, precision shielding is used in COVID-19 management, and requires estimates of mass infection in key groups. As a result, rapid tests for the virus could be a useful screening tool for asymptomatic virus shedders who are about to come into contact with sensitive groups. In Africa and other low- and middle-income countries there is high rate of COVID-19 under-diagnosis, due to the high cost of molecular assays. Exploring alternate assays to the reverse transcriptase polymerase chain reaction (RT-PCR) for COVID-19 diagnosis is highly warranted. Aim: This review explored the feasibility of using alternate molecular, rapid antigen, and serological diagnostic assays to accurately and precisely diagnose COVID-19 in African populations, and to mitigate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR diagnostic challenges in Africa. Method: We reviewed publications from internet sources and searched for appropriate documents available in English. This included Medline, Google Scholar, and Ajol. We included primary literature and some review articles that presented knowledge on the current trends on SARS-CoV-2 diagnostics in Africa and globally. Results: Based on our analysis, we highlight the utility of four different alternatives to RT-PCR. These include two isothermal nucleic acid amplification assays (loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA)), rapid antigen testing, and antibody testing for tackling difficulties posed by SARS-CoV-2 RT-PCR testing in Africa. Conclusion: The economic burden associated COVID-19 mass testing by RT-PCR will be difficult for low-income nations to meet. We provide evidence for the utility and deployment of these alternate testing methods in Africa and other LMICs.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of ‘sample-in and result-out’ with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/μL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/μL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/μL with a total assay time of 15–30 min.

Rapid and specific detection of mango (Mangifera indica) in processed food using an isothermal nucleic acid amplification assay

Mango (Mangifera indica) is one of the most popular tropical fruits around the world. It is also widely and commonly used in many cuisines and products in the food industry. However, the anaphylactic reaction caused by mango has long been reported as a major problem for consumption in recent years. To prevent allergens in mango, the best way is to avoid mango in the diet. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of mango in food. Four specifically designed LAMP primers targeting the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions were used to address the LAMP reaction for amplifying mango DNA. The results demonstrated that the detection of mango DNA was specifically validated by the LAMP primer. The sensitivity of LAMP for detecting mango DNA is equivalent to that of the traditional PCR method. The LAMP primer sets showed high specificity for detecting the DNA of mango and had no cross-reactions to other species. Moreover, when mango was mixed with other fruits at different ratios, no cross-reactivity for the detection of mango DNA was manifested during LAMP. Finally, genomic DNAs extracted from different heat-processed mangos were used as templates; the detection of mango DNA by LAMP was not significantly affected and was reproducible. As to this established LAMP herein, mango ingredients can be detected, and commercial foods containing mango can also be identified. This assay will be useful and have potential for the rapid detection of mango DNA in practical food markets.

On-Chip DNA Analysis of Tuberculosis Based on Magnetic Nanoparticle Clustering Induced by Rolling Circle Amplification Products

We present on-chip implementation of an isothermal nucleic acid amplification assay with optomagnetic detection. Isothermal rolling circle amplification (RCA) was designed for the highly specific detection of a single point mutation in the subsequence of the katG catalase peroxidase gene responsible for resistance against the antibiotic isoniazid in Mycobacterium tuberculosis . The assay was integrated using streptavidin-coated magnetic microbeads (MMBs) as a movable substrate to transport the synthetic katG target and products between the sequential processing steps. RCA products were detected via optomagnetic measurements of the binding of functionalized magnetic nanoparticles (MNPs). Binding of MNPs to RCA products leads to an increase of their hydrodynamic size and consequently a shift of the optomagnetic signal to lower frequencies. We first analyzed the affinity of DNA probes used for target capture on MMBs and binding of MNPs to RCA products, respectively. This enabled us to choose conditions where amplification products were released from the MMBs and where their binding to MNPs was optimal. The optimized assay was transferred to a polymer chip with three connected chambers filled with reaction buffers for capture, RCA, and detection. The chip was operated in a setup integrating temperature control, transport of the DNA on magnetic carriers, and dose-dependent detection of MNP clustering. For a total assay time of about 2 h, we demonstrate specific detection of the antibiotic resistance in a synthetic tuberculosis DNA target with a detection limit of 4 pM.

Validation of the Listeria Right NowTM Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment.

Background: Listeria Right NowTM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested MethodSM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation

Zika virus has only recently gained attention due to recent large outbreaks worldwide. An easy to use nucleic acid amplification test could play an important role in the early detection of the infection and patient management. Here, we report a rapid and robust isothermal nucleic acid amplification assay for the detection of Zika virus. The method is cost-effective and compatible with portable instrumentation, enabling near patient testing and field use.

Evaluation of the Alere i Influenza A&B assay for rapid identification of influenza A and influenza B viruses.

The Alere i Influenza A&B assay is a novel isothermal nucleic acid amplification assay capable of detecting and differentiating influenza A and B viruses in approximately 15 min with minimal hands-on time. This study was conducted in order to assess the performance of the Alere i Influenza A&B assay compared to molecular techniques, considered to be gold standard methods, to evaluate the results. A total of 119 nasopharyngeal swabs collected from inpatients with influenza-like illness were included in the study using both archived and prospectively collected samples from adults and children. Prospectively collected samples were also compared to the Alere BinaxNOW® Influenza A & B Card. The overall sensitivity for detection of influenza A and B viruses compared to those of molecular techniques were 65.96 % and 53.33 % respectively, while the specificity was 98.51 % and 95.96 %. Compared to the Alere BinaxNOW® Influenza A & B Card, the Alere i assay is considerably more sensitive for detection of influenza A and B viruses, although both tests demonstrated excellent specificity for diagnosis of influenza viruses.

Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification.

Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

Validation of the ANSR® Listeria Method for Detection of Listeria spp. in Selected Foods.

ANSR® Listeria was previously certified as Performance Tested Method(SM) 101202 for detection of Listeria spp. on selected environmental surfaces. This study proposes a matrix extension to the method for detection of Listeria spp. in selected food matrixes. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in less than 50 min, requiring only simple instrumentation. Inclusivity testing was performed using a panel of 51 strains of Listeria spp., representing the species L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, and L. welshimeri. All strains tested were detected by the ANSR assay. Exclusivity testing of 30 strains representing non-Listeria Gram-positive bacteria yielded no evidence of cross-reactivity. Performance of the ANSR method for detection of Listeria spp. was compared to that of reference culture procedures for pasteurized liquid egg, pasteurized 2% milk, Mexican-style cheese, ice cream, smoked salmon, lettuce, cantaloupe, and guacamole. Data obtained in these unpaired studies and analyzed using a probability of detection model demonstrated that there were no statistically significant differences in results between the ANSR and reference culture methods, except for milk at 16 h and cantaloupe. In milk and smoked salmon, ANSR sensitivity was low at 16 h and therefore the recommended incubation time is 24 h. In cantaloupe, ANSR was found to be more sensitive than the reference culture method at both 16 and 24 h in independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in selected food types.

Detection of influenza A and B with the Alere ™ i Influenza A & B: a novel isothermal nucleic acid amplification assay.

BackgroundRapid influenza diagnostic tests (RIDTs) have an important role in clinical decision-making; however, the performances of currently available assays vary widely.ObjectivesWe evaluated the performance of the Alere™ i Influenza A&B (Alere™ iNAT), a rapid isothermal nucleic acid amplification assay that has recently received FDA clearance, for the detection of influenza A and B viruses during the Australian influenza season of 2013. Results were compared to two other RIDTs tested in parallel; Quidel Sofia® Influenza A+B fluorescent immunoassay (FIA) and Alere™ BinaxNOW® Influenza A & B immunochromatographic (ICT) assay.MethodsA total of 202 paired nasopharyngeal swabs collected from patients ≥16 years old with an influenza-like illness (ILI) were eluted in 2 ml of universal transport medium (UTM) that was used to perform all three RIDTs in parallel. Reverse-transcription polymerase chain reaction (RT-PCR) was used as the reference standard.ResultsCompared to RT-PCR, Alere™ iNAT detected 77·8% influenza A positive samples versus 71·4% and 44·4% for the Quidel Sofia® Influenza A+B FIA and BinaxNOW® Influenza A & B ICT assay, respectively. For influenza B, Alere™ iNAT detected 75% of those positive by RT-PCR, versus 33·3% and 25·0% for Sofia® and BinaxNOW®, respectively. The specificity of Alere™ iNAT was 100% for influenza A and 99% for influenza B.ConclusionsAlere™ i Influenza A&B is a promising new rapid influenza diagnostic assay with potential point-of-care applications.

Validation of the ANSR® Listeria Method for Detection of Listeria spp. in Environmental Samples

ANSR Listeria is a new diagnostic assay for detection of Listeria spp. in sponge or swab samples taken from a variety of environmental surfaces. The method is an isothermal nucleic acid amplification assay based on the nicking enzyme amplification reaction technology. Following single-step sample enrichment for 16-24 h, the assay is completed in 40 min, requiring only simple instrumentation. In inclusivity testing, 48 of 51 Listeria strains tested positive, with only the three strains of L. grayi producing negative results. Further investigation showed that L. grayi is reactive in the ANSR assay, but its ability to grow under the selective enrichment conditions used in the method is variable. In exclusivity testing, 32 species of non-Listeria, Gram-positive bacteria all produced negative ANSR assay results. Performance of the ANSR method was compared to that of the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of Listeria spp. in sponge or swab samples taken from inoculated stainless steel, plastic, ceramic tile, sealed concrete, and rubber surfaces. Data were analyzed using Chi-square and probability of detection models. Only one surface, stainless steel, showed a significant difference in performance between the methods, with the ANSR method producing more positive results. Results of internal trials were supported by findings from independent laboratory testing. The ANSR Listeria method can be used as an accurate, rapid, and simple alternative to standard culture methods for detection of Listeria spp. in environmental samples.

A Handheld Point-of-Care Genomic Diagnostic System

The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.

Instrument-free exothermic heating with phase change temperature control for paper microfluidic devices.

Many infectious diseases, as well as some cancers, that affect global health are most accurately diagnosed through nucleic acid amplification and detection. There is a great need to simplify nucleic acid-based assay systems for use in global health in low-resource settings as well as in settings that do not have convenient access to laboratory staff and equipment such as doctors' offices and home care settings. In developing countries, unreliable electric power, inadequate supply chains, and lack of maintenance for complex diagnostic instruments are all common infrastructure shortfalls. Many elements of instrument-free, disposable, nucleic acid amplification assays have been demonstrated in recent years. However, the problem of instrument-free, low-cost, temperature-controlled chemical heating remains unsolved. In this paper we present the current status and results of work towards developing disposable, low-cost, temperature-controlled heaters designed to support isothermal nucleic acid amplification assays that are integrated with a two-dimensional paper network. Our approach utilizes the heat generated through exothermic chemical reactions and controls the heat through use of engineered phase change materials to enable sustained temperatures required for nucleic acid amplification. By selecting appropriate exothermic and phase change materials, temperatures can be controlled over a wide range, suitable for various isothermal amplification methods, and maintained for over an hour at an accuracy of +/- 1°C.

Printing the Next Generation of Point-of-Care Diagnostic Tests

Two-dimensional paper networks (2DPNs) area new set of devices allow complex chemical processing in a very low-cost format. We have, for the last 5 years, been learning how to translate what we have learned about point-of-care diagnostic technologies in conventional microfluidics into the language of porous media. The wicking of fluids in porous materials (like paper, nitrocellulose membranes, etc.) allows us to discard pumps, which permits great savings in complexity and cost, and the potential to perform complex tests without any permanent instruments. However, there are many physical and chemical differences between open ducts and porous media. We have put a good deal of effort into understanding the performance and design rules of simple paper systems. Furthermore, application of reagents to the papers in liquid and dry form is central to device development, testing, and economical manufacture. Currently, the primary applications for this technology in our lab are highlysensitive multiplexed immunoassays and multiplexed isothermal nucleic acid amplification assays. All assays are designed with visible optical readout that can be captured and quantified using camera-equipped cellular phones.

Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

BackgroundSignal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus.ResultsHere we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis.ConclusionThe SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.