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A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection.

ABSTRACT: A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (Tm) Molecular Beacon DNA probes. A positive-temperature-coefficient heater with a temperature sensor for real-time thermal regulation was integrated into the control unit, which generated actuation signals for droplet manipulation. To enhance the specificity of the LAMP reaction, low-Tm Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 μL of LAMP reaction samples containing purified Trypanosoma brucei DNA were required, which represented over a 10x reduction of reagent consumption when comparing with the conventional off-chip LAMP. On-chip LAMP for unknown sample detection could be accomplished in 40 min with a detection limit of 10 copies/reaction. Also, we accomplished an on-chip melting curve analysis of the Molecular Beacon probe from 30 to 75 °C within 5 min, which was 3x faster than using a commercial qPCR machine. Discrimination of non-specific amplification and lower risk of aerosol contamination for on-chip LAMP also highlight the potential utilization of this system in clinical applications. The entire platform is open for further integration with sample preparation and fluorescence detection towards a total-micro-analysis system.

SUBMITTER: Wan L

PROVIDER: S-EPMC5673945 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The rapid and accurate diagnostic methods to identify carbapenemase-producing organisms (CPO) is of great importance for controlling the CPO infection. Herein, we have developed a microfluidic chip-based technique to detect CPO and assessed its clinical value in detecting CPO directly from blood cultures (BCs). The detection performance of the microfluidic chip-based LAMP amplification method was analyzed retrospectively on a collection of 192 isolates including molecularly characterized 108 CPO and 84 non-CPO and prospectively on a collection of 133 positive BCs with or without CPO suspicion, respectively. In the retrospective study, the microfluidic chip-based LAMP amplification method exhibited 87.5% accuracy (95% CI [82.0-91.5]), 97.7% sensitivity (95% CI [91.2-99.6]), 78.8% specificity (95% CI [69.5-86.0]), 79.6% positive predictive value (PPV) (95% CI [70.6-86.5]) and 97.6% negative predictive value (NPV) (95% CI [90.9-99.6]). Among the 192 isolates, 22 (11.5%) false-positives (FP) and 2 (1.0%) false negatives (FN) were observed. In the prospective study, the 133 routine isolates of positive BCs including 18 meropenem-resistant CPO and 115 non-CPO were assessed, and 4 FP were observed in non-CPO and CPO, respectively. The current method showed a total detection performance of 94.0% accuracy (95% CI [88.4-97.1]), 100.0% sensitivity (95% CI [73.2-100.0]), 93.2% specificity (95% CI [86.7-96.8]), 63.6% PPV (95% CI [40.8-82.0]) and 100.0% NPV (95% CI [95.8-100.0]). In summary, the microfluidic chip-based LAMP amplification method is reliable for the rapid screening and detection of CPO with high accuracy, sensitivity, and specificity, and could easily be implemented in clinical microbiology laboratories. IMPORTANCE Rapid and accurate identification of CPO may reduce the genetic exchanges among bacteria and prevent further dissemination of carbapenemases to non-CPO. The current method had designed microfluidic chip-based LAMP amplification method for multiplex detection of carbapenemase genes and evaluated the detection performance of the newly method. The current method can rapidly screen and detect CPO with high accuracy, sensitivity, and specificity, and could easily be implemented in clinical microbiology laboratories, as this will reduce the carbapenem resistance issues worldwide.

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A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection.

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