Highly Sensitive Detection of Genomic DNA Using Rapid Real-Time PCR Based on Oscillating Flow
Authors: Nagai Hidenori , Furutani Shunsuke 
Published: December 2024

Some individuals are diagnosed with urethritis owing to the detection of gonorrhoea or chlamydia infection through clinical tests; however, there are cases in which neither is detected despite exhibiting symptoms. One of the causative bacteria of non-chlamydial non-gonococcal urethritis is Mycoplasma genitalium, and real-time polymerase chain reaction (qPCR), an accurate examination of causative bacteria for proper drug administration, has been used for its detection. However, given that qPCR is time-consuming, applying it to same-day testing is difficult. Therefore, a rapid qPCR system was developed using a microfluidic device.

The microfluidic device used for rapid thermal cycling with oscillating flow in each detection unit was 76 mm × 52 mm × 4 mm in size. The PCR solution was injected into the microchannel from the inlet. The microfluidic device, including the liquid plug, was placed on the chip holder, and three small heaters were embedded into the rapid qPCR system. The temperature of each heater was constantly controlled for reverse transcription (optionally), DNA denaturation and ex-tension by DNA polymerase for PCR. Furthermore, both ends of the microchannel were automatically connected with pneumatic lines using the needles installed in the upper cover of the chip holder when pushed down. This system employed a pair of microblowers as the driving force for the reciprocating liquid transfer. When the microblower was turned off, it quickly released the pressure between the liquid plug and microblower without additional valves, such that the liquid transfer in the microchannel could be stopped at any position immediately. The liquid plug of the PCR solution was reciprocally transferred at programmed timing by switching the two microblowers controlled by the main unit. The detection unit also had an array of three fluorescence detectors aligned along the microchannel. To detect the passage of the PCR solution, the fluorescence of fluorescein mixed with the PCR solution was measured in the middle of the two heaters for denaturation and extension. The fluorescence signal was used to precisely control the stop position on the heater. The remaining two fluorescence detectors were located above the two heaters individually and were used for liquid position detection and evaluation of the amplification of the MgPa gene and internal control.

A standard curve was evaluated against gDNA standards of M. genitalium. For the thermal cycle conditions, heating at 96˚C for 10 s, 3 s at 96˚C, and 6 s at 59˚C was repeated 50 times. A dilution series of 0.063 to 5.0×104 copies/run was used as the gDNA concentration of M. genitalium. In addition, to confirm the detection frequency at low concentrations, samples were tested nine times at concentrations where the presence or absence of gDNA in the PCR solution varied statistically. Fig. (a) shows the detection frequency at each concentration of gDNA of M. genitalium. The rapid RT-PCR system was amplified from extremely low concentrations of samples even if the number was < 1 copy/run, although the frequency was not 100%. Fig. (b) shows the standard curve obtained from these amplification curves, which shows good linearity up to 5 copies/run. The PCR efficiency was calculated to be 96.3%. For concentrations of < 5 copies, the success or failure of detection was speculated to depend on the stochastic inclusion of the template DNA in the PCR solution. Fig. (c) shows the detection frequency for each gDNA concentration of M. genitalium and a fitting curve based on the cumulative distribution function (CDF) of the Poisson distribution. The expected value of the maximum likelihood CDF corresponded to 1 copy/run, which would theoretically yield the best LOD. The LOD with a 95% confidence interval (CI) was estimated to be 3 copies/run from this fitting curve. The statistically estimated limit of detection was equivalent to the amplification from a single molecule of genomic DNA, and the PCR efficiency was calculated to be almost 100%. Moreover, the analysis time for a negative judgement obtained without amplification was within 11 min. Thus, the developed rapid RT-PCR system would be useful for the same-day testing of infectious bacteria.

Figure 1