Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

Kim, Jung Sun; Jung, Seungwon; Byoun, Mun Sub; Yoo, Changhoon; Sim, Sang Jun; Lim, Chae Seung; Kim, Sung Woo; Kim, Sang Kyung

doi:10.1371/journal.pone.0190451

Cited by 8 publications

(6 citation statements)

References 26 publications

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“…As the number of target genes has rapidly increased, assay multiplicity has become essential in diagnostics. ,, Although multiplex assays are typically achieved using different color channels of the fluorescence, it has been difficult to use multiple fluorescence channels across wavelengths to avoid interference from the light sources of photonic heating . pPIN qPCR is ideal for expanding the number of targets without concerns about spectral overlap because the targets are distinguished by geometric codes on pPINs corresponding to the target gene.…”

Section: Results and Discussionmentioning

confidence: 99%

Ultrafast Real-Time PCR in Photothermal Microparticles

Kim

Lee

Park³

et al. 2022

ACS Nano

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As the turnaround time of diagnosis becomes important, there is an increasing demand for rapid, point-ofcare testing (POCT) based on polymerase chain reaction (PCR), the most reliable diagnostic tool. Although optical components in real-time PCR (qPCR) have quickly become compact and economical, conventional PCR instruments still require bulky thermal systems, making it difficult to meet emerging needs. Photonic PCR, which utilizes photothermal nanomaterials as heating elements, is a promising platform for POCT as it reduces power consumption and process time. Here, we develop a photonic qPCR platform using hydrogel microparticles. Microparticles consisting of hydrogel matrixes containing photothermal nanomaterials and primers are dubbed photothermal primer-immobilized networks (pPINs). Reduced graphene oxide is selected as the most suitable photothermal nanomaterial to generate heat in pPIN due to its superior light-to-heat conversion efficiency. The photothermal reaction volume of 100 nL (predefined by the pPIN dimensions) provides fast heating and cooling rates of 22.0 ± 3.0 and 23.5 ± 2.6 °C s −1 , respectively, enabling ultrafast qPCR within 5 min only with optical components. The microparticle-based photonic qPCR facilitates multiplex assays by loading multiple encoded pPIN microparticles in a single reaction. As a proof of concept, four-plex pPIN qPCR for bacterial discrimination are successfully demonstrated.

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Section: Results and Discussionmentioning

confidence: 99%

Ultrafast Real-Time PCR in Photothermal Microparticles

Kim

Lee

Park³

et al. 2022

ACS Nano

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“…Furthermore, the identification of malarial parasite species is critical for appropriate treatment [ 30 , 31 , 32 ]. Efforts are being made to develop practical diagnosis methods to control malaria, such as microscopy-based, rapid diagnostic tests (RDTs), and techniques to detect nucleic acids specific to each species that are highly sensitive and specific [ 33 , 34 , 35 ].…”

Section: Resultsmentioning

confidence: 99%

Multiplex Assay for Rapid Detection and Analysis of Nucleic Acid Using Barcode Receptor Encoded Particle (BREP)

Jung

Bong

Na³

2022

Biomedicines

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Several multiplex nucleic acid assay platforms have been developed in response to the increasing importance of nucleic acid analysis, but these assays should be optimized as per the requirements of point-of-care for clinical diagnosis. To achieve rapid and accurate detection, involving a simple procedure, we propose a new concept in the field of nucleic acid multiplex assay platforms using hydrogel microparticles, called barcode receptor-encoded particles (BREPs). The BREP assay detects multiple targets in a single reaction with a single fluorophore by analyzing graphically encoded hydrogel particles. By introducing sets of artificially synthesized barcode receptor and barcode probes, the BREP assay is easily applicable in multiplexing any genetic target; sets of barcode receptors and barcode probes should be designed delicately for universal application. The performance of the BREP assay was successfully verified in a multiplex assay for the identification of different malaria species with high sensitivity, wide dynamic range, fast detection time, and multiplexibility.

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“…The primers are normally immobilized onto different materials depending on the technique and type of assay. 51 , 52 As usual in any development of a diagnostic tool, the sensing platform is first validated with standard spiked samples before experiments are done with real samples (i.e., swabs, blood, serum, and plasma samples). 3 , 53 The primers for RT-PCR and LAMP assays are produced by molecular biology methods.…”

Section: Sars-cov-2 Diagnosismentioning

confidence: 99%

“…These are the cases of RT-PCR and LAMP assays. The primers are normally immobilized onto different materials depending on the technique and type of assay. , As usual in any development of a diagnostic tool, the sensing platform is first validated with standard spiked samples before experiments are done with real samples (i.e., swabs, blood, serum, and plasma samples). , The primers for RT-PCR and LAMP assays are produced by molecular biology methods. In the immunoassays, the biorecognition elements are frequently proteins (biomarkers) which will bind specifically to antibodies immobilized onto the sensing platform. ,, These devices can be applied to a large number of samples, as blood, serum, plasma, urine, and saliva.…”

Section: Sars-cov-2 Diagnosismentioning

confidence: 99%

On the Challenges for the Diagnosis of SARS-CoV-2 Based on a Review of Current Methodologies

et al. 2020

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Diagnosis of COVID-19 has been challenging owing to the need for mass testing and for combining distinct types of detection to cover the different stages of the infection. In this review, we have surveyed the most used methodologies for diagnosis of COVID-19, which can be basically categorized into genetic-material detection and immunoassays. Detection of genetic material with real-time polymerase chain reaction (RT-PCR) and similar techniques has been achieved with high accuracy, but these methods are expensive and require time-consuming protocols which are not widely available, especially in less developed countries. Immunoassays for detecting a few antibodies, on the other hand, have been used for rapid, less expensive tests, but their accuracy in diagnosing infected individuals has been limited. We have therefore discussed the strengths and limitations of all of these methodologies, particularly in light of the required combination of tests owing to the long incubation periods. We identified the bottlenecks that prevented mass testing in many countries, and proposed strategies for further action, which are mostly associated with materials science and chemistry. Of special relevance are the methodologies which can be integrated into point-of-care (POC) devices and the use of artificial intelligence that do not require products from a well-developed biotech industry.

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Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

Cited by 8 publications

References 26 publications

Ultrafast Real-Time PCR in Photothermal Microparticles

Ultrafast Real-Time PCR in Photothermal Microparticles

Multiplex Assay for Rapid Detection and Analysis of Nucleic Acid Using Barcode Receptor Encoded Particle (BREP)

On the Challenges for the Diagnosis of SARS-CoV-2 Based on a Review of Current Methodologies

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