Real-time capillary convective PCR based on horizontal thermal convection

Qiu, Xianbo; Shu, Jung I.; Baysal, Oktay; Wu, Jie; Qian, Shizhi; Ge, Shengxiang; Li, Ké; Ye, Xiangzhong; Xia, Ningshao; Yu, Duli

doi:10.1007/s10404-019-2207-0

Cited by 22 publications

(14 citation statements)

References 19 publications

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“…Water bath-based PCR has been abandoned because of its low degree of automation, low amplification efficiency, and lack of consistency ( Mahanama and Wilson-Davies, 2021 ). Air-heated-based real-time PCR has subsequently been developed with air as a heat-transfer medium ( Qiu et al, 2019 ). Although this method increases the amplification efficiency to a certain extent because of the low thermal conductivity and specific heat capacity of air ( Miao et al, 2020 ), additional energy consumption and cost were increased.…”

Section: Introductionmentioning

confidence: 99%

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules

Lin

Song

Shao³

et al. 2022

Front. Bioeng. Biotechnol.

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Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

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Section: Introductionmentioning

confidence: 99%

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules

Lin

Song

Shao³

et al. 2022

Front. Bioeng. Biotechnol.

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“…For a properly designed CPCR reactor, a consistent temperature field on the reactor must be established to achieve the stable and repeatable thermal convection for robust amplification [19,20]. Compared to conventional PCR, which normally takes 1-2 h, CPCR can be performed within 30 min, which is competitive in POC diagnostics [21][22][23][24]. Recently, few microfluidic CPCR NAAT systems with complicated sample preparation have been reported [25], which nevertheless demonstrate their potentiality in POC diagnostics with simple heating and ultra-fast amplification.…”

Section: Introductionmentioning

confidence: 99%

An Integrated, Real-Time Convective PCR System for Isolation, Amplification, and Detection of Nucleic Acids

Miao

Guo

et al. 2022

Chemosensors

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Convective PCR (CPCR) can perform rapid nucleic acid amplification by inducing thermal convection to continuously, cyclically driving reagent between different zones of the reactor for spatially separate melting, annealing, and extending in a capillary tube with constant heating temperatures at different locations. CPCR is promoted by incorporating an FTA membrane filter into the capillary tube, which constructs a single convective PCR reactor for both sample preparation and amplification. To simplify fluid control in sample preparation, lysed sample or wash buffer is driven through the membrane filter through centrifugation. A movable resistance heater is used to heat the capillary tube for amplification, and meanwhile, a smartphone camera is adopted to monitor in situ fluorescence signal from the reaction. Different from other existing CPCR systems with the described simple, easy-to-use, integrated, real-time microfluidic CPCR system, rapid nucleic acid analysis can be performed from sample to answer. A couple of critical issues, including wash scheme and reaction temperature, are analyzed for optimized system performance. It is demonstrated that influenza A virus with the reasonable concentration down to 1.0 TCID50/mL can be successfully detected by the integrated microfluidic system within 45 min.

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“…Convective PCR thermal cycling, in which PCR is conducted by inducing spontaneous thermal convection inside a capillary tube, is a promising tool due to its simple heating strategy and low cost. However, the non-uniformity of the temperature is still a challenge, because the sample unit is extremely sensitive to temperature fluctuations [ 19 ]. Photonic PCR, which is based on the photothermal effects of nanomaterials (e.g., gold nanoparticles, magnetic nanoparticles, and graphene), is another strategy to enhance the heating rate of thermal cycling.…”

Section: Introductionmentioning

confidence: 99%

Conductive Silver/Carbon Fiber Films for Rapid Detection of Human Coronavirus

et al. 2022

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Polymerase chain reaction has gained attention since the outbreak of novel coronavirus in 2019. Due to its high specificity and capability for early detection, it is considered a standard method for the diagnosis of infectious diseases. However, the conventional thermocyclers used for nucleic acid amplification are not suitable for point-of-care testing applications, as they require expensive instruments, high-power consumption, and a long turnaround time. To suppress the widespread of the pandemic, there is an urgent need for the development of a rapid, inexpensive, and portable thermal cycler. Therefore, in this paper, we present a conductive silver/carbon fiber film-based thermal cycler with low power consumption (<5 W), efficient heating (~4.5 °C/s), low cost (<USD 200), and handheld size (11.5 × 7.1 × 7.5 mm). The conductive film, which was used as a heating source of the thermal cycler, was fabricated by the electrochemical deposition method. The successful coating of Ag was characterized by a scanning electron microscope and confirmed by energy-dispersive X-ray spectroscopy. The film showed excellent electrical/thermal conductivity and durability. Using our thermal cycler, 35 cycles of amplification were accomplished within 10 min. We also successfully demonstrated the multiplexed detection of various human coronaviruses (e.g., OC43, 229E, and NL63) using our thermal cycler.

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Real-time capillary convective PCR based on horizontal thermal convection

Cited by 22 publications

References 19 publications

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules

Performance Evaluation of a Novel Ultrafast Molecular Diagnostic Device Integrated With Microfluidic Chips and Dual Temperature Modules

An Integrated, Real-Time Convective PCR System for Isolation, Amplification, and Detection of Nucleic Acids

Conductive Silver/Carbon Fiber Films for Rapid Detection of Human Coronavirus

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