A thermally baffled device for highly stabilized convective PCR

Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, P. K.; Su, Chen; Jeng, Chien-Chung

doi:10.1002/biot.201100453

Cited by 69 publications

(55 citation statements)

References 11 publications

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“…according to the default signal-to-noise (S/N) thresholds. 4,17 To determine analytical sensitivity of the established assay, a synthetic plasmid containing the T7 promoter-LTR/ gag region of the FIV genome cassette e was used to prepare RNA transcribed in vitro (IVT) RNA using a commercial kit.…”

Section: Research-article2015mentioning

confidence: 99%

Rapid and sensitive detection ofFeline immunodeficiency virususing an insulated isothermal PCR-based assay with a point-of-need PCR detection platform

et al. 2015

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Abstract. Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5′ long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform-a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats.

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Section: Research-article2015mentioning

confidence: 99%

Rapid and sensitive detection ofFeline immunodeficiency virususing an insulated isothermal PCR-based assay with a point-of-need PCR detection platform

et al. 2015

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“…[12][13][14] This temperature gradient induces a continuous, sustained circulation of reactants between hot and cool zones of the reaction, thus stratifying the reaction into spatially separate and stable melting, annealing, and extension zones. [15][16][17][18][19] With thermal convection, convective PCR thus eliminates the need for controlled time-domain temperature cycling and external pumps or other fluid actuation or flow control. [20][21][22][23][24] However, most of existed capillary convective PCR (CCPCR) systems are unavailable for commercial application because of those unresolved issues related to repeatability, reliability, convenience, and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Characterization and analysis of real-time capillary convective PCR toward commercialization

et al. 2017

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Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single-and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and photodiode, and the real-time fluorescence detection method is compared with the postamplification end-point detection method based on a dipstick assay. Thirdly, to reduce the system complexity, e.g., to simplify parameter tuning of the feedback control, an internal-model-control-based proportional-integral-derivative controller is adopted for accurate temperature control. Fourth, as a proof of concept, CCPCR with pre-loaded dry storage of reagent inside the capillary PCR tube is evaluated to better accommodate to point-of-care diagnosis. The critical performances of improved CCPCR, especially with sensitivity, repeatability, and reliability, have been thoroughly analyzed with different experiments using influenza A (H1N1) virus as the detection sample. Published by AIP Publishing. [http://dx

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“…It was designed to target two of the most conserved regions found in the ORF6 and ORF7 genes in the PRRSV genome [23]. Furthermore, in iiPCR, natural liquid convection established in a capillary tube can cycle the reaction components sequentially through different temperature zones to achieve the 3 stages (denaturation, annealing, and extension) of PCR [26][27][28]. Consequently, the annealing step is not done at a fixed temperature in iiPCR, allowing primers and probes to bind to sequences with minor mismatches [29].…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by a Duplex Reverse Transcription Insulated Isothermal PCR on a Field-Deployable System

Kuo¹,

Lo²,

Chen³

et al. 2017

J Vet Sci Technol

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Citation: Kuo HC, Lo DY, Chen CL, Lee CH, Shen YH, et al. (2017) AbstractPorcine reproductive and respiratory syndrome virus (PRRSV) is an important porcine pathogen globally. Reverse transcription-polymerase chain reaction (RT-PCR) for PRRSV detection is an important tool for disease management and control. Clinical sensitivity of RT-PCR for PRRSV detection is compromised to a certain degree by the high genetic diversity in the PRRSV genome. A duplex RT-insulated isothermal PCR (RT-iiPCR) for the North America lineage of PRRSV (PRRSV-NA) has been developed by targeting both ORF6 and ORF7 to increase test inclusivity. In this study, its limit of detection 95% was determined to be about 5 genome equivalents per reaction by testing a serial dilution of in-vitro transcribed RNA. The PRRSV-NA duplex RT-iiPCR was compared with an ORF7 real-time RT-PCR (rRT-PCR) published previously for the evaluation of analytical and clinical performance. Both tests did not react with seven common swine pathogens. The two methods had similar detection endpoints for viral RNA of two PRRSV-NA isolates. Further tests with 187 swine samples showed that 14 of the 90 rRT-PCR-negative and 2 of the 97 rRT-PCR-positive samples were positive and negative by the duplex RT-iiPCR, respectively. The two methods had 91.44% agreement (95% confidential interval: 87.26 -95.62%, κ=0.83). Repeat testing could not resolve 13 of the discrepant samples (all negative by rRT-PCR and positive by RT-iiPCR). Further RT-nested PCR analysis and DNA sequencing analysis of the ORF7 region supported that the target RNA was present in these samples. Therefore, the PRRSV-NA duplex RT-iiPCR appeared to have higher clinical sensitivity than the reference rRT-PCR. Working on a field-deployable device, the PRRSV-NA duplex RT-iiPCR has potential to serve as a fast and sensitive tool for PRRSV detection at points of need.

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A thermally baffled device for highly stabilized convective PCR

Cited by 69 publications

References 11 publications

Rapid and sensitive detection ofFeline immunodeficiency virususing an insulated isothermal PCR-based assay with a point-of-need PCR detection platform

Rapid and sensitive detection ofFeline immunodeficiency virususing an insulated isothermal PCR-based assay with a point-of-need PCR detection platform

Characterization and analysis of real-time capillary convective PCR toward commercialization

Rapid Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by a Duplex Reverse Transcription Insulated Isothermal PCR on a Field-Deployable System

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