iLOCS

Development of a laptop-sized mobile molecular-diagnostic, integrated lab-on-a-chip (iLOCS) system for the rapid molecular diagnostic of nosocomial infections

G. Dame1 ; M. Hügle 1,2; G. A. Urban 2 ; F.T. Hufert 1
1Institute of Microbiology and Virology, MHB, Medical School Brandenburg, Germany
2 University of Freiburg, IMTEK, Chair for Sensors, Germany

Background

Clostridium difficile (C. diff.) associated diarrhea are of growing concern in seriously ill patients after antibiotic treatment. Norovirus infection represents a frequent and central hygiene management problem especially in geriatric care.

We focussed on the development of a mobile diagnostic point-of-care system (POCTs) which allows rapid on-site screening in cases with severe diarrhea. Based on nucleic acids detection, we are aiming at the development of a laptop-sized mobile molecular diagnostic integrated lab-on-a-chip system (iLOCS). The two diagnostically important gastrointestinal infections (Norovirus, C. difficile) will be detected by an isothermal recombinase polymerase amplification (RPA) assay, which allows genetic amplification within 15 minutes. Thus, the system should deliver a reliable result below 30 minutes. Here we present the results of two RPA assays for molecular detection of Norovirus and C. difficile.

Furthermore, we show the first results of our RPA-based Lap-on-Chip development. In the future iLOCS can be extended to a number of other pathogens or any other gene which today is detected by real-time PCR. C. difficile. Darüber hinaus präsentieren wir die ersten Ergebnisse unserer Ingenieurarbeiten zur RPA-basierten Lab-on-Chip-Entwicklung. In Zukunft soll iLOCS auf RPA-Basis auch zur Diagnostik weiterer Krankheitserreger eingesetzt werden und könnte bisherige diagnostische PCR-Verfahren ersetzen.

Methods

Based on gene bank genetic analysis, qPCR and RPA primer/probe sets were generated for C. diff. (Tox A and B) and Norovirus (GGI, GGII). Primer sets were for PCR were analysed on a LightCycler 480 and RPA primer sets were investigated by an RPA reader (Tube scanner). To determine the sensitivity of the assays a Probit analysis was carried out with eight different runs using our synthetic RNA or DNA standards.

Norovirus

Figure 1. Analytical sensitivity as determined on RNA run-off transcripts of Norovirus (8 runs) Norovirus GGI sensitivity: 10 1 -10 2 genomes/µl

Results
Molecular diagnostic assays.

RPA assays for Norovirus and toxin B of C. difficile were developed. All RPA assays showed a specificity of almost 100%. The Norovirus and C. difficile B toxin B RPA assays showed a sensitivity of at least 102 genomes/µl each. The Norovirus RPA results are shown in Figure 1. These results are comparable to standard real-time PCR assays. Using our RPA standards, the Lab-on-Chip was tested for RPA suitability. Results were compared to the Tube scanner reference system as shown in Figure 2.

vergleich

Figure 2. Comparison of C. difficile RPA using the standard RPA reader (Tubescanner, red line) with the LabChip system (blue line) based on a defined number of genome copies.

iLOCS development

For the construction of iLOCS a chip was developed to purify and concentrate nucleic acids using two integrated platinum electrodes and three hydrogels. Furthermore, we constructed a power supply unit as well as a chip holder shown in Figure 3.

Conclusion

The aim of this approach was the development of a molecular detection system for the rapid detection of norovirus and toxin-bearing Clostridium difficile using an isothermal amplification system. Such a system could be widely used in outpatient and inpatient medical care. Rapid and reliable diagnostic tools in a sample-to-result format can reduce the workload in diagnostic facilities, minimize hands-on-time and make an easy on-site testing at home possible. A data transfer option to an eHealth Center for further processing would allow real-time medical validation. POCT molecular diagnostics systems offer also new opportunities for on-site diagnosis of infectious diseases (ID) in countries with low health care standards, in any type of field missions or outbreak investigation. Integrated in a suitcase-lab it offers an easy and affordable tool for rapid molecular ID diagnosis in low resource settings.

Mikrofluidischer

Figure 3. (Left) Microfluidic chip for preconcentration and lysis of bacteria as well as gel-electrophoresis of nucleic acids and a CNC milled custom chip holder (Right).

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