Mikidache Madi

Development and Initial Results of a Low Cost, Disposable, Point-of-Care Testing Device for Pathogen Detection

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ~90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tubes port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ~1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus.

Development and evaluation of multiplex RT-LAMP assays for rapid and sensitive detection of foot-and-mouth disease virus

This paper describes the evaluation of four novel real-time multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for rapid and sensitive diagnosis of foot-and-mouth disease (FMD). In order to overcome the genetic diversity of FMD viruses (FMDV), these multiplex RT-LAMP assay pairs were established by combining four newly designed primer sets with two primer sets that had been previously published. Using a real-time turbidimeter to detect amplification products and a panel of 300 samples collected throughout the world over a 78-year period, the performance of the multiplex RT-LAMP assays was compared with a FMDV-specific real-time RT-PCR assay. The most successful of the four multiplex RT-LAMP assays achieved a diagnostic sensitivity and specificity of 98.0% and 98.1%, and did not falsely detect FMDV in known negatives or samples containing swine vesicular disease virus, vesicular stomatitis virus or vesicular exanthema of swine virus. Furthermore, the analytical sensitivity of this multiplex RT-LAMP assay was at least as good as the individual component RT-LAMP tests. This is the first report of the development of a multiplex RT-LAMP to accommodate the high sequence variability encountered in RNA virus genomes and these results support the use of RT-LAMP as a cost-effective tool for simple diagnosis of FMD.

Rapid detection of foot-and-mouth disease virus using a field-portable nucleic acid extraction and real-time PCR amplification platform.

Rapid and accurate field diagnostic tools can be used to support clinical diagnosis during outbreaks of exotic livestock diseases. This study evaluated a mobile PCR amplification platform that performs RNA extraction, real-time reverse-transcription PCR (rRT-PCR) and interpretation of results without operator intervention. Initial studies showed that there was equivalence between the detection limit generated by RNA extracted using the mobile platform and an automated laboratory-based system. In subsequent studies, two validated laboratory-based foot-and-mouth disease virus (FMDV)-specific rRT-PCRs were transferred onto the mobile platform and all assay steps (RT incubation and PCR amplification) were performed with non-lyophilised reagents using an optimised protocol of less than 60 min. The limit of detection of the rRT-PCR on the mobile PCR platform was equivalent to an automated laboratory-based assay used for routine diagnosis of FMDV and there was concordance between these methods for results generated using samples in a reference laboratory proficiency panel. Future studies will be directed at the development and validation of commercially-viable consumables using lyophilised PCR reagents for FMDV and the evaluation of this technology in FMD endemic countries using field samples.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Summary Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot‐and‐mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple‐to‐use technologies, including molecular‐based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)‐specific reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) and real‐time RT‐PCR (rRT‐PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory‐based rRT‐PCR. However, the lack of robust ‘ready‐to‐use kits’ that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT‐PCR and RT‐LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real‐time, and for the RT‐LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Detection of Capripoxvirus DNA Using a Field-Ready Nucleic Acid Extraction and Real-Time PCR Platform.

Summary Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real‐time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory‐based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory‐based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory‐based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings.

Development of a non-infectious encapsidated positive control RNA for molecular assays to detect foot-and-mouth disease virus

Highlights • FMDV is highly infectious and can only be handled in high-containment laboratories.• This study has developed encapsidated control particles containing FMDV RNA.• The construct contains target sequences for molecular assays used to detect FMDV.• These control particles were evaluated using routine tests used for FMD diagnosis.• These particles are non-infectious and temperature-stable.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Summary Highly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT‐PCR diagnostic system (Enigma MiniLab®) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real‐time RT‐PCR (rRT‐PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: Optimisation and evaluation of a high-throughput screening method with potential for disease surveillance

Highlights • FMDV was detected by rRT-PCR in milk up to 28 days post contact challenge.• FMDV was detected in milk collected from infected farms in the field (UK 2007).• FMDV detection was possible when a milk sample was diluted up to 10−7 in negative milk.• Pooled milk has the potential to be a valuable sample type for FMDV surveillance.

Genome Sequences of Nine Vesicular Stomatitis Virus Isolates from South America

ABSTRACT We report nine full-genome sequences of vesicular stomatitis virus obtained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species (clade III), while two isolates belonged to the Indiana serotype/species.