Rapid Isothermal Amplification Research Articles

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Biosensor-based multiple cross displacement amplification platform for visual and rapid identification of hepatitis C virus.

Hepatitis C remains a global health problem, especially in poverty-stricken areas. A rapid and sensitive point-of-care (POC) diagnostic tool is critical for the early detection and timely treatment of hepatitis C virus (HCV) infection. Here, for the first time, we reported a novel molecular diagnostic assay, termed reverse transcription multiple cross displacement amplification integrated with a gold-nanoparticle-based lateral flow biosensor (RT-MCDA-AuNPs-LFB), which was developed for rapid, sensitive, specific, and visual identification of HCV. HCV-RT-MCDA induced rapid isothermal amplification through a specific primer set targeting the 5'untranslated region gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a that are prevalent in China. The optimal reaction temperature and time for RT-MCDA-AuNPs-LFB were 68°C and 25 min, respectively. The limit of detection of the assay was 10 copies per test, and the specificity was 100% for the experimental strains. The whole detection procedure, including crude nucleic acid isolation (~5 min), RT-MCDA (68°C, 25 min), and visual AuNPs-LFB result confirmation (less than 2 min), was performed within 35 min. The preliminary results indicated that the HCV-RT-MCDA-AuNPs-LFB assay could be a valuable tool for sensitive, specific, visual, cost-saving, and rapid detection of HCV and has potential as a POC diagnostic platform for field screening and early clinical detection of HCV infection.

FTA-LAMP based biosensor for a rapid in-field detection of Globodera pallida-the pale potato cyst nematode.

The combination of a sensitive and specific magnetoresistive sensing device with an easy DNA extraction method and a rapid isothermal amplification is presented here targeting the on-site detection of Globodera pallida, a potato endoparasitic nematode. FTA-cards were used for DNA extraction, LAMP was the method developed for DNA amplification and a nanoparticle functionalized magnetic-biosensor was used for the detection. The combinatorial effect of these three emerging technologies has the capacity to detect G. pallida with a detection limit of one juvenile, even when mixed with other related species. This combined system is far more interesting than what a single technology can provide. Magnetic biosensors can be combined with any DNA extraction protocol and LAMP forming a new solution to target G. pallida. The probe designed in this study consistently distinguished G. pallida (∆Vac binding/Vac sensor above 1%) from other cyst nematodes (∆Vac binding/Vac sensor below 1%). It was confirmed that DNA either extracted with FTA-cards or Lab extraction Kit was of enough quantity and quality to detect G. pallida whenever present (alone or in mixed samples), ensuring probe specificity and sensitivity. This work provides insights for a new strategy to construct advanced devices for pathogens in-field diagnostics. LAMP runs separately but can be easily integrated into a single device.

Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.

453 Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2.

Human transmission of SARS-CoV-2 and emergent variants of concern continue to occur globally, despite mass vaccination campaigns. Public health strategies to reduce virus spread should therefore rely, in part, on frequent screening with rapid, inexpensive, and sensitive tests. We evaluated two digitally integrated rapid tests and assessed their performance using stored nasal swab specimens collected from individuals with or without COVID-19. An isothermal amplification assay combined with a lateral flow test had a limit of detection of 10 RNA copies per reaction, and a positive percent agreement (PPA)/negative percent agreement (NPA) during the asymptomatic and symptomatic phases of 100%/100% and 95.83/100%, respectively. Comparatively, an antigen-based lateral flow test had a limit of detection of 30,000 copies and a PPA/NPA during the asymptomatic and symptomatic phases of 82.86%/98.68% and 91.67/100%, respectively. Both the isothermal amplification and antigen-based lateral flow tests had optimized detection of SARS-CoV-2 during the peak period of transmission; however, the antigen-based test had reduced sensitivity in clinical samples with qPCR Ct values greater than 29.8. Low-cost, high-throughput screening enabled by isothermal amplification or antigen-based techniques have value for outbreak control.

Rapid isothermal amplification of several kilo base pair linear DNA

Polymerase Chain Reaction (PCR) is an essential method used to amplify or copy nucleic acids. PCR requires a thermal cycler to alternate between strand separation at high temperatures and replication/copying at low temperature. We eliminated thermal cycling and kept all desired PCR features in place through enabling a new SSB (Single-Stranded Binding protein) and helicase assisted isothermal amplification method named SHARP (SSB-Helicase Assisted Rapid PCR). SHARP uses the same starting set of primers and template DNA as PCR does, carries out the reaction at a constant temperature, and outputs the same amplicon as PCR with lengths of up to 6000 base pairs within 5 to 30 minutes, a feature no existing isothermal amplification method can match.

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

Research Note: Development of rapid isothermal amplification assay for detection of duck circovirus

Duck circovirus disease (DuCVD) caused by duck circovirus (DuCV) continues to spread in recent years, which brings serious harm to the poultry industry, so early diagnosis of DuCVD is of great significance for the prevention and control of this disease. Specific primers and probes for DuCV were designed in this study. Reverse primers and probes were modified at the 5′ ends with biotin and fluorescein, respectively, and they were combined with dipsticks labeled with biotin antibodies and fluorescein antibodies to establish a recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assay for detection of duck circovirus. By using this method, the reaction products reached detectable levels in about 20 min as a result of rapid amplification at a constant temperature of 37℃. The detection results could be observed by dripping the reaction products onto the dipstick within 2 to 3 min. The RAA-LFD method has good specificity and high sensitivity (102 copies/μL). Compared with conventional polymerase chain reaction (PCR), RAA-LFD has no power limit on the testing instrument, and is easy to use, saving more time and manpower, so it is more suitable for clinical detection.

Rapid Isothermal Amplification for the Buccal Detection SARS-CoV-2 in the Context of Out-Patient COVID-19 Screening.

A commercially available isothermal amplification of SARS-CoV-2 RNA was applied to self-collected saliva samples using dry dental cotton rolls, which were held in the mouth for two minutes. Of 212 tests, isothermal amplification yielded three (0.14%) invalid results, 120 (56.6%) positive results and 89 (42%) negative results. Compared to reference RT-PCR assays routinely performed simultaneously on nasopharyngeal swabs, excluding the three invalid isothermal amplification assays and one RT-PCR invalid assay, these figures indicated that 119/123 (96.7%) samples were positive in both methods and 85/85 samples were negative in both methods. Four positive buccal swabs which were missed by the isothermal amplification, exhibited Ct values of 26–34 in reference RT-PCR assays. Positive isothermal amplification detection was achieved in less than 10 min. Supervision of the self-sampling procedure was key to achieve these performances. These data support the proposal to use the protocol reported in this paper, including supervised buccal self-sampling, to screen people suspected of having COVID-19 at the point of care.

Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si–OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies/mL for sample input volumes of up to 600 μL, and can be performed in less than 1 h from sample collection to result readout. This assay demonstrated a 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.

First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Rapid isothermal amplification and portable detection system for SARS-CoV-2.

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.

Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype‐based identification. The CRISPR‐Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR‐Cas13a platform SHERLOCK (Specific High‐sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of “proof of principle” experiments to characterize SHERLOCK’s ability to accurately, sensitively and rapidly distinguish three fish species of management interest co‐occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK’s ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction‐free DNA collection protocol, as well as the option of instrument‐free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV.

Recombinase Polymerase Amplification (RPA) for the Rapid Isothermal Detection of Spongospora subterranea f. sp. subterranea and Potato Mop-Top Virus

The amplification of specific nucleic acid sequences with high specificity and sensitivity is an essential technique for pathogen detection. Recombinase polymerase amplification (RPA) is a rapid isothermal amplification method. Here, we demonstrate the end-point and real-time detection of Spongospora subterranea f. sp. subterranea (Sss) using RPA and potato mop-top virus (PMTV) using reverse transcription (RT)-RPA. Oligonucleotide primers were designed for amplification that target the internal transcribed spacer 1 (ITS1) region and the coat protein readthrough (CP-RT) domain for the detection of Sss and PMTV, respectively. Our data showed that real-time RPA can detect 100 of Sss sporosori per gram of soil, while real-time RT-RPA could detect in ~1 ng total RNA of the PMTV-infected tuber. For Sss detection, the R2 value for real-time RPA and real-time PCR was 98.0% by linear regression analysis in the concentration range of 100–34,000 sporosori per gram of soil. The developed RPA assay here may provide a useful alternative tool for the rapid, simple and reliable detection of Sss and PMTV in diagnostic laboratories and in-field testing.

Comparison of loop-mediated isothermal amplification (LAMP) and cross-priming amplification (CPA) for detection of African swine fever virus.

The reliable and rapid diagnosis of infectious animal diseases presents an exceptionally im- portant aspect when considering their control and prevention. The paper describes the compara- tive evaluation of two rapid isothermal amplification methods for diagnosis of African swine fever (ASF). The robustness of loop-mediated isothermal amplification (LAMP) and the cross-priming amplification (CPA) were compared using samples obtained from ASF confirmed animals. Both assays were evaluated in order to define their diagnostic capabilities in terms of ASF diagnosis and reproducibility of the results. Investigations showed no cross-reactivity for other pig patho- gens and no significant differences in the specificity of both assays. The sensitivity of LAMP reached 90%, while that of CPA was 70%. In conclusion, both methods are suitable for imple- mentation in preliminary ASF diagnosis but further improvements are required to enhance their diagnostic sensitivity.

Antarctic thermolabile uracil-DNA-glycosylase-supplemented multiple cross displacement amplification using a label-based nanoparticle lateral flow biosensor for the simultaneous detection of nucleic acid sequences and elimination of carryover contamination

Here, we report a novel and universal methodology, termed “Antarctic thermolabile uracil-DNA-glycosylase (AUDG)-supplemented nucleic acid amplification techniques (NAAs) using a labeled-based nanoparticle lateral flow biosensor (LFB)” (AUDG-NAAs-LFB), which merges enzymatic (AUDG) digestion of contaminant amplicons with different nucleic acid amplification techniques (NAAs), and uses a lateral flow biosensor (LFB) for the rapid and visual confirmation of the presence of a target nucleic acid sequence. AUDG-NNAs-LFB is a one-pot, closedvessel assay, that can effectively eliminate false-positive signals arising from either carryover contaminants or the interaction between labeled primers. A new LFB was devised for detecting three targets (two amplicons generated from amplification of target sequences, and a chromatography control), without the need for probe-hybridization or additional incubation steps. As a proof of concept, multiple cross displacement amplification (MCDA), which is a specific, sensitive, and rapid isothermal amplification method, was selected as the model amplification technique to demonstrate the feasibility of AUDG-NAAs-LFB. As a result, we demonstrate the applicability of the AUDG-MCDA-LFB method for simultaneously detecting high-risk human papillomaviruses genotypes 16 and 18, which are the most and second-most prevalent strains of the virus reported in women worldwide. We also confirm the principle behind the AUDG-MCDA-LFB assay and validate its sensitivity, reproducibility, and specificity using serial dilutions of the type-specific plasmids, as well as clinical samples. This proof-of-concept method (AUDG-MCDA-LFB) can be easily reconfigured to detect various nucleic acid sequences by redesigning the specific MCDA primers.

Ebolavirus diagnosis made simple, comparable and faster than molecular detection methods: preparing for the future

BackgroundThe 2014/2015 Ebolavirus outbreak resulted in more than 28,000 cases and 11,323 reported deaths, as of March 2016. Domestic transmission of the Guinea strain associated with the outbreak occurred mainly in six African countries, and international transmission was reported in four countries. Outbreak management was limited by the inability to rapidly diagnose infected cases. A further fifteen countries in Africa are predicted to be at risk of Ebolavirus outbreaks in the future as a consequence of climate change and urbanization. Early detection of cases and reduction of transmission rates is critical to prevent and manage future severe outbreaks. We designed a rapid assay for detection of Ebolavirus using recombinase polymerase amplification, a rapid isothermal amplification technology that can be combined with portable lateral flow detection technology. The developed rapid assay operates in 30 min and was comparable with real-time TaqMan™ PCR.MethodsDesigned, screened, selected and optimized oligonucleotides using the NP coding region from Ebola Zaire virus (Guinea strain). We determined the analytical sensitivity of our Ebola rapid molecular test by testing selected primers and probe with tenfold serial dilutions (1.34 × 1010− 1.34 × 101 copies/μL) of cloned NP gene from Mayinga strain of Zaire ebolavirus in pCAGGS vector, and serially diluted cultured Ebolavirus as established by real-time TaqMan™ PCR that was performed using ABI7500 in Fast Mode. We tested extracted and reverse transcribed RNA from cultured Zaire ebolavirus strains – Mayinga, Gueckedou C05, Gueckedou C07, Makona, Kissidougou and Kiwit. We determined the analytical specificity of our assay with related viruses: Marburg, Ebola Reston and Ebola Sudan. We further tested for Dengue virus 1–4, Plasmodium falciparum and West Nile Virus (Kunjin strain).ResultsThe assay had a detection limit of 134 copies per μL of plasmid containing the NP gene of Ebolavirus Mayinga, and cultured Ebolavirus and was highly specific for the Zaire ebolavirus species, including the Guinea strain responsible for the 2014/2015 outbreak. The assay did not detect related viruses like Marburg, Reston, or Sudan viruses, and other pathogens likely to be isolated from clinical samples.ConclusionsOur assay could be suitable for implementation in district and primary health laboratories, as only a heating block and centrifuge is required for operation. The technique could provide a pathway for rapid screening of patients and animals for improved management of outbreaks.