Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR
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 dev...
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| Format: | Article |
| Language: | English |
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MDPI AG
2025-01-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/15/1/36 |
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| author | Xiaoqing Dai Meng Cao Zunliang Wang |
| author_facet | Xiaoqing Dai Meng Cao Zunliang Wang |
| author_sort | Xiaoqing Dai |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-b20ea8b66e1946bfa1f52d5292536c9f |
| institution | Kabale University |
| issn | 2079-6374 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj-art-b20ea8b66e1946bfa1f52d5292536c9f2025-01-24T13:25:30ZengMDPI AGBiosensors2079-63742025-01-011513610.3390/bios15010036Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCRXiaoqing Dai0Meng Cao1Zunliang Wang2State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Si Pai Lou 2, Nanjing 210096, ChinaState Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Si Pai Lou 2, Nanjing 210096, ChinaState Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Si Pai Lou 2, Nanjing 210096, ChinaWe 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.https://www.mdpi.com/2079-6374/15/1/36multiplex detectiondroplet digital PCRdigital melting curve analysisfluorescence imagingmolecular diagnostics |
| spellingShingle | Xiaoqing Dai Meng Cao Zunliang Wang Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR Biosensors multiplex detection droplet digital PCR digital melting curve analysis fluorescence imaging molecular diagnostics |
| title | Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR |
| title_full | Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR |
| title_fullStr | Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR |
| title_full_unstemmed | Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR |
| title_short | Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR |
| title_sort | digital melting curve analysis for multiplex quantification of nucleic acids on droplet digital pcr |
| topic | multiplex detection droplet digital PCR digital melting curve analysis fluorescence imaging molecular diagnostics |
| url | https://www.mdpi.com/2079-6374/15/1/36 |
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