Rapid nested-PCR for tyrosinase gene detection on chip

Sciancalepore, Anna Giovanna; Polini, Alessandro; Mele, Elisa; Girardo, Salvatore; Cingolani, R.; Pisignano, Dario

doi:10.1016/j.bios.2010.09.008

Cited by 21 publications

(12 citation statements)

References 34 publications

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“…On the other hand, the microchip system took only 40 min which is 70% less time compared to the thermocycler system. This 40-min runtime is comparable to Sciancalepore et al's microfluidic device which took 46.5 min [16]. The thermocycler system that was used in this study was considered to be an older model.…”

Section: Nested Pcrmentioning

confidence: 96%

“…Nested PCR was first successfully demonstrated in this close-loop PCR system. Although nested PCR had been demonstrated in Sciancalepore et al's oscillatory flow PCR system [16], the fundamental working mechanism was different. Hence, the future implementation of nPCR on chip will also be different.…”

Section: Nested Pcrmentioning

confidence: 99%

“…Hence, at the end, only half of the protocol was done on chip. Sciancalepore et al demonstrated nested PCR for tyrosinase gene detection on their oscillatory flow microchip system [16], in which the PCR mixture was embedded in oil phase, moved back and forth, oscillating through three temperature zone. Successful PCR runs were achieved by passing this microchip for first PCR run and then again for the second PCR run.…”

Section: Introductionmentioning

confidence: 99%

“…PCR on chip system can be implemented in the form of well-based or continuous-flow [13]. In continuous-flow PCR system, it could be further sub-categorized to closed-loop [14], fixedloop [15] and oscillatory [16]. Although the first PCR on chip was introduced by Northrup et al in 1993 [17], nested PCR protocol was rarely implemented in LOC related literatures.…”

Section: Introductionmentioning

confidence: 99%

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Nested PCR in magnetically actuated circular closed-loop PCR microchip system

et al. 2012

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We demonstrate a new and sensitive amplification technique (referred to as Nested Polymerase Chain Reaction; nPCR). It based on a magnetically actuated circular closed-loop PCR microchip system. nPCR involves the use of two sets of primers in two successive PCR runs, and allows the amplification of a single locus from a minute quantity of template DNA. Two sets of primers are specially designed to a target 500-bp region of the bacteriophage lambda template DNA in the first PCR run, and a 247-bp region of the targeted 500-bp first PCR product in the second PCR run. PCR is run on the microchip system and concurrently in regular thermocycler for comparison. The products are analyzed by conventional agarose gel electrophoresis. The detection limit for the initial template DNA is 1.63×10 5 copies per μL (or 8.67 pg) for the first PCR run, and 1.63 copies per μL (or 0.0867 fg) for the second run. The results are comparable to a regular thermocycler. This preliminary study opens a new gateway to future development of specialized nPCR on chip.

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Section: Nested Pcrmentioning

confidence: 96%

Section: Nested Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nested PCR in magnetically actuated circular closed-loop PCR microchip system

et al. 2012

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“…Each substrate material has different properties and therefore different advantages and disadvantages. It should be noted that the commercially available glass capillary has been used to produce the glass-PDMS hybrid chip for the oscillatory-flow PCR (Chen et al 2007b;Polini et al 2010;Sciancalepore et al 2011). Compared with the all-PMDS chip, this hybrid configuration provides greatly reduced water evaporation, ease and low cost of fabrication, and conformal contact with the heater elements (Polini et al 2010).…”

Section: Introductionmentioning

confidence: 97%

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

Zhang

Wang

Xing

2011

Biomed Microdevices

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In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10(-2) ng/μl (278-bp, S. enterica), 11.2 × 10(-2) ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10(-2) ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10(4) copies/μl, 3.58 × 10(4) copies/μl, and 1.79 × 10(4) copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.

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Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology

et al. 2016

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On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

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Rapid nested-PCR for tyrosinase gene detection on chip

Cited by 21 publications

References 34 publications

Nested PCR in magnetically actuated circular closed-loop PCR microchip system

Nested PCR in magnetically actuated circular closed-loop PCR microchip system

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology

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