Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR

Abstract

We present a cost-effective and simple multiplex nucleic acid quantification method using droplet digital PCR (ddPCR) with digital melting curve analysis (MCA). This approach eliminates the need for complex fluorescent probe design, reducing both costs and dependence on fluorescence channels. We developed a convolutional neighborhood search algorithm to correct droplet displacement during heating, ensuring precise tracking and accurate extraction of melting curves. An experimental protocol for digital MCA on the ddPCR platform was established, enabling accurate quantification of six target pathogen genes using a single fluorescence channel, with an average accuracy of 85%. Our method overcomes the multiplexing limitations of ddPCR, facilitating its application in multi-target pathogen detection.

Keywords: digital melting curve analysis; droplet digital PCR; fluorescence imaging; molecular diagnostics; multiplex detection.

Figure 1

Schematic diagram of the digital PCR melting curve analysis, with details as follows: (A) A tube of nucleic acid sample is prepared for testing. (B) The sample is distributed into approximately 20,000 microdroplets of 0.8 nL, each using vibration injection technology on the ddPCR platform. (C) A total of 40 cycles of PCR amplification are performed for each droplet. (D) After the amplification is complete, the fluorescence images of the microdroplet array are captured. (E) A series of droplet array images are captured during heating. (F) The melting peak profiles of positive droplets can be obtained by taking the negative derivative of the melting curves, which are derived from the changes in the fluorescence signals from these droplets. (G) The positive droplets within the target’s Tm range are counted, which, along with the derived negative droplet count, allows for target concentration quantification using the Poisson distribution model.

Figure 2

Structure and functional modules of the ddPCR platform used in this study. The platform is composed of three key modules: droplet generation, fluorescence collection, and heating. The droplet generation module employs an interfacial emulsification method driven by injection vibration technology to generate uniform water-in-oil droplet array. The fluorescence collection module captures the fluorescence signals from the amplified droplets, while the heating module regulates the temperature for PCR cycling and digital MCA.

Figure 3

Schematic diagram (A) and flowchart (B) of the convolutional neighborhood search algorithm for droplet displacement correction.

Figure 4

Protocol for digital MCA on the ddPCR platform.

Figure 5

Fluorescence images of droplet arrays, each containing a single target gene fragment: (A) cap5F, (B) iucD, (C) lytA, (D) atoE, (E) uidA, and (F) yfkN, captured at different temperatures.

Figure 6

Droplet tracking through the micro-displacement correction method. The droplets (indicated by white arrows) at 60 °C (A) are accurately localized after minor displacement at 90 °C (B). Correctly identified droplets are highlighted by red circles.

Figure 7

The original digital melting curve (A) after droplet digital PCR and its corresponding characteristic melting peak (B), derived from discrete differentiation of the original curve. The high-resolution melting curve (C) generated from RT-qPCR and its characteristic melting peak (D), calculated through discrete differentiation of the original high-resolution melting curve.

Figure 8

Digital melting curves (A–G: left) and corresponding melting peak profiles (A–G: right) of nucleic acid fragments within all positive droplets for 2- to 6-plex ddPCR assays. Different colored bars indicate the melting peaks for six target nucleic acid sequences: red for cap5F, blue for atoE, yellow for uidA, green for yfkN, purple for lytA, and cyan for iucD.

Figure 9

Quantitative accuracy of multiplex nucleic acid detection based on digital MCA: (A) Linear correlation between target gene concentrations calculated from multiplex quantification and reference template concentrations. (B) Relationship between multiplex quantitative accuracy and the number of target genes.