Labordaten

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Labordaten

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

Dame G.1, Hügle M.1,2, Urban G. A. 2 and Hufert F.T.1,

1 Institute of Microbiology and Virology, MHB, Medical School Brandenburg, Germany

2 University of Freiburg, IMTEK, Chair for Sensors, Germany

Background

Norovirus is one of the most frequent cause of acute infectious gastroenteritis in Europe. In addition, the number of C. difficile infection (CDI) is increasing and of growing concern in seriously ill patients after antibiotic treatment. The clinical spectrum of CDI ranges from mild diarrhea to severe life-threatening pseudomembranous colitis. Both pathogens cause severe problems in the hygiene management of hospitals and in any type of shared facilities. Early rapid diagnostic is crucial to prevent pathogen dissemination and to stop the outbreak.

Based on isothermal gene amplification using the recombinase polymerase amplification technique (RPA) we are aiming at the development of a laptop-sized mobile molecular diagnostic integrated lab-on-a-chip system (iLOCS) for the rapid diagnostic of infectious diseases. Compared to standard real-time PCR assays RPA yields a result within 5-15 minutes (standard real-time PCR 1-2 h) using low-energy consuming isothermal amplification.

Here we present the results of two RPA assays for the rapid molecular detection of Norovirus and C. difficile. Furthermore, we show the first engineering results of our RPA-based Lap-on-a-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.

Methods

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

Results

Molecular diagnostic assays. RPA assays for Norovirus and toxin B of C. difficile were developed. All RPA assays showed a specificity of 100%. The Norovirus and C. difficile 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. In addition the Lab-on-a-Chip was tested for RPA suitability using our RPA standards. The results were compared to the Tube scanner reference system as shown in Figure 2.

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 specific power supply unit as well as a chip holder shown in Figure 3.

Conclusion

The aim of this project 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 to stop Norovirus outbreaks and C. difficile infections.

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.

Figure 1. Analytical sensitivity as determined on RNA run-off transcripts of Norovirus (8 runs)

Norovirus GGI sensitivity: 10 1 -10 2 genomes/µl

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.

figure3

Figure 3. (Left) Microfluidic chip for preconcentration and bacterial lysis and for downstream nucleic acid purification using gel electrophoresis. (Right) CNC milled custom chip holder.

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