Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens

Abstract

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.

Figure 1

Microfluidic PCR setup. Left: The self contained instrument including power supply, control electronics, fluorimeter module, heaters and cartridge deck, which is shown with the cartridge loaded (arrow). Center: Photograph of assembled microfluidic cartridge comprising an 86 × 86 mm PCB chip, polymer spacer/gasket and glass top-plate with drilled holes. Right: Schematic of PCR chip showing electrode positions relative to heaters, magnets and detectors.

Figure 2

Limit of detection and precision testing performed with simulated clinical samples on (A) the conventional real-time PCR platform and (B) the microfluidic real-time PCR platform. Concentration versus mean CT with standard deviations shown.