A fully integrated duplex RT-LAMP device for the detection of viral infections.

Respiratory viruses can cause epidemics or pandemics, which are worldwide outbreaks of disease. The severity of these events varies depending on the virus, its characteristics, along with environmental factors. The frequency of epidemics and pandemics caused by respiratory viruses is difficult to predict, but the potential severity of such events underlines the importance of continued monitoring, research, and preparation for emerging infectious diseases. To help improve pandemic preparedness, we created a fully integrated duplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) device targeting two respiratory viruses, influenza A/X-31 virus and bovine coronavirus, as a replacement for SARS-CoV-2. This device can be adapted to any other respiratory virus. In this study, we showed and evaluated a prototype of a microfluidic system, and showed that duplex RT-LAMP can detect and distinguish between the two viruses, with LoDs of 2,000 copies/ml for bovine coronavirus and 200 copies/ml for influenza A/X-31 virus.

Self-Sampled Gargle Water Direct RT-LAMP as a Screening Method for the Detection of SARS-CoV-2 Infections.

We assessed the viability of self-sampled gargle water direct RT-LAMP (LAMP) for detecting SARS-CoV-2 infections by estimating its sensitivity with respect to the gold standard indirect RT-PCR of paired oro-nasopharyngeal swab samples. We also assessed the impact of symptom onset to test time (STT)-i.e., symptom days at sampling, on LAMP. In addition, we appraised the viability of gargle water self-sampling versus oro-nasopharyngeal swab sampling, by comparing paired indirect RT-PCR results. 202 oro-nasopharyngeal swab and paired self-sampled gargle water samples were collected from hospital patients with COVID-19 associated symptoms. LAMP, indirect and direct RT-PCR were performed on all gargle water samples, and indirect RT-PCR was performed on all oro-nasopharyngeal samples. LAMP presented a sensitivity of 80.8% (95% CI: 70.8-90.8%) for sample pairs with sub-25 Ct oro-nasopharyngeal indirect RT-PCR results, and 77.6% (66.2-89.1%) sensitivity for sub-30 Ct samples with STT ≤ 7 days. STT, independently of Ct value, correlated negatively with LAMP performance. 80.7% agreement was observed between gargle water and oro-nasopharyngeal indirect RT-PCR results. In conclusion, LAMP presents an acceptable sensitivity for low Ct and low STT samples. Gargle water may be considered as a viable sampling method, and LAMP as a screening method, especially for symptomatic persons with low STT values.

DjinniChip: evaluation of a novel molecular rapid diagnostic device for the detection of Chlamydia trachomatis in trachoma-endemic areas.

BACKGROUND: The clinical signs of active trachoma are often present in the absence of ocular Chlamydia trachomatis infection, particularly following mass drug administration. Treatment decisions following impact surveys and in post-control surveillance for communities are currently based on the prevalence of clinical signs, which may result in further unnecessary distribution of mass antibiotic treatment and the increased spread of macrolide resistance alleles in 'off-target' bacterial species. We therefore developed a simple, fast, low cost diagnostic assay (DjinniChip) for diagnosis of ocular C. trachomatis for use by trachoma control programmes. METHODS: The study was conducted in the UK, Germany and Tanzania. For clinical testing in Tanzania, specimens from a sample of 350 children between the ages of 7 to 15 years, which were part of a longitudinal cohort that began in February 2012 were selected. Two ocular swabs were taken from the right eye. The second swab was collected dry, kept cool in the field and archived at - 80 °C before sample lysis for DjinniChip detection and parallel nucleic acid purification and detection/quantification by qPCR assay. RESULTS: DjinniChip was able to reliably detect > 10 copies of C. trachomatis per test and correctly identified 7/10 Quality Control for Molecular Diagnostics C. trachomatis panel samples, failing to detect 3 positive samples with genome equivalent amounts ≤ 10 copies. DjinniChip performed well across a range of typical trachoma field conditions and when used by lay personnel using a series of mock samples. In the laboratory in Tanzania, using clinical samples the sensitivity and specificity of DjinniChip for C. trachomatis was 66% (95% CI 51-78) and 94.8 (95% CI 91-97%) with an overall accuracy of 90.1 (95% CI 86.4-93). CONCLUSIONS: DjinniChip performance is extremely promising, particularly its ability to detect low concentrations of C. trachomatis and its usability in field conditions. The DjinniChip requires further development to reduce inhibition and advance toward a closed system. DjinniChip results did not vary between local laboratory results and typical trachoma field settings, illustrating its potential for use in low-resource areas to prevent unnecessary rounds of MDA and to monitor for C. trachomatis recrudescence.

Detection and identification of Staphylococcus aureus using magnetic particle enhanced surface plasmon spectroscopy.

In this work, an approach for SPR spectroscopy using the liSPR system is examined that combines signal amplification by PCR and magnetic nanoparticles in one injection step. Therefore, the synthesis of PCR products was performed on the beads similar to a solid-phase PCR, termed PCR-on-a-bead. The functionality of this PCR was proven using an enzymatic assay. For validation the detection of oligonucleotides by SPR, an asymmetric PCR product was investigated. A signal increase upon binding of the PCR product to the specific probes was observed. In addition, surface regeneration of the chip was examined and reuse for at least two times ascertained. Amplification of the SPR signal by magnetic beads was verified but no signal was detected for PCR products immobilized on particles prior to injection.

An integrated versatile lab-on-a-chip platform for the isolation and nucleic acid-based detection of pathogens.

AIM: Processing of the samples in molecular diagnostics is complex and labor-intensive. An integrated and automated platform for sample preparation and nucleic acid-based detection can significantly relieve this burden for the users. RESULTS: We present a prototype of a versatile and integrated platform for the detection of pathogens in various liquid media. We describe a proof-of-concept for the integrated isolation of bacteria, cell lysis with optional DNA extraction, DNA amplification and detection in two different reactions, loop-mediated isothermal amplification and PCR, on a single microfluidic platform. CONCLUSION: The platform enables the transition from large sample volume to microfluidic format. The design and open interface enable its versatile application for various nucleic acid-based assays, from simple to complex setups.

Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein.

Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4 × 10(6)-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.

Application of nanotechnology in miniaturized systems and its use for advanced analytics and diagnostics - an updated review.

A combination of Micro-Electro-Mechanical Systems and nanoscale structures allows for the creation of novel miniaturized devices, which broaden the boundaries of the diagnostic approaches. Some materials possess unique properties at the nanolevel, which are different from those in bulk materials. In the last few years these properties became a focus of interest for many researchers, as well as methods of production, design and operation of the nanoobjects. Intensive research and development work resulted in numerous inventions exploiting nanotechnology in miniaturized systems. Modern technical and laboratory equipment allows for the precise control of such devices, making them suitable for sensitive and accurate detection of the analytes. The current review highlights recent patents in the field of nanotechnology in microdevices, applicable for medical, environmental or food analysis. The paper covers the structural and functional basis of such systems and describes specific embodiments in three principal branches: application of nanoparticles, nanofluidics, and nanosensors in the miniaturized systems for advanced analytics and diagnostics. This overview is an update of an earlier review article.

Application of nanotechnology in miniaturized systems and its use in medical and food analysis.

A combination of Micro-Electro-Mechanical Systems and nanoscale structures allows for the creation of novel miniaturized devices, which broaden the boundaries of the diagnostic approaches. Some materials possess unique properties at the nanolevel, which are different from those in bulk materials. In the last years these properties became a focus of interest for many researchers, as well as methods of production, design and operation of the nanoobjects. Intensive research and development work resulted in numerous inventions, exploiting nanotechnology in miniaturized systems. Modern technical and laboratory equipment allows for the precise control of such devices, making them suitable for sensitive and accurate detection of the analytes. The current review highlights recent patents in the field of nanotechnology in microdevices, applicable for medical and food analysis. The paper covers the structural and functional basis of such systems and describes specific embodiments in three principal branches: application of nanoparticles, nanofluidics, and nanosensors in the miniaturized systems for advanced analytics and diagnostics.