A novel fluorescence quantification method for polymerase chain reaction system

Chien, Jui Hung; Lee, Da Sheng; Cheng, Yi Ting; Yeh, Shou Huei; Chou, Wen Ping; Chen, Ping‐Hei

doi:10.1016/j.optcom.2006.05.043

Cited by 2 publications

(4 citation statements)

References 9 publications

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“…The 150-bp detection gene for HCV is used. The detection limit of the circulating PCR chip is found to be 10 copies/μL; L 100-bp DNA markers; C5 10 5 copies/μL; C4 10 4 copies/μL; C3 10 3 copies/μL; C2 10 2 copies/μL; C1 10 copies/μL (Chien et al 2006). The intensity of the fluorescence signals from a standard DNA marker (Yeastern Biotech Corp., Taiwan) and the amplified DNA products were compared to quantify the amplified DNA products.…”

Section: Resultsmentioning

confidence: 95%

“…A forward primer (5′-GTT GAT CCA AGA AAG GAC CCG GTC-3′), and a reverse primer (5′-GAG GAA CTA CTG TCT TCA CG-3′) were used (Chien et al 2006). The PCR mixture has the following composition: 7.6 μL of ddH 2 O; 2.4 μL of 25 mM Mg 2+ ; 2 μL of 2.5 μM of forward primer and 2 μL of 5.0 μM of reverse primer; 2 μL of enzyme mix (Roche, USA), and 4 μL of HCV DNA template with different initial concentrations.…”

Section: Virus Strain and Pcr Reagentmentioning

confidence: 99%

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A micro circulating PCR chip using a suction-type membrane for fluidic transport

Chien

Wang

Hsieh

et al. 2008

Biomed Microdevices

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A new micromachined circulating polymerase chain reaction (PCR) chip is reported in this study. A novel liquid transportation mechanism utilizing a suction-type membrane and three microvalves were used to create a new microfluidic control module to rapidly transport the DNA samples and PCR reagents around three bio-reactors operating at three different temperatures. When operating at a membrane actuation frequency of 14.29 Hz and a pressure of 5 psi, the sample flow rate in the microfluidic control module can be as high as 18 microL/s. In addition, an array-type microheater was adopted to improve the temperature uniformity in the reaction chambers. Open-type reaction chambers were designed to facilitate temperature calibration. Experimental data from infrared images showed that the percentage of area inside the reaction chamber with a thermal variation of less than 1 degrees C was over 90% for a denaturing temperature of 94 degrees C. Three array-type heaters and temperature sensors were integrated into this new circulating PCR chip to modulate three specific operating temperatures for the denaturing, annealing, and extension steps of a PCR process. With this approach, the cycle numbers and reaction times of the three separate reaction steps can be individually adjusted. To verify the performance of this circulating PCR chip, a PCR process to amplify a detection gene (150 base pairs) associated with the hepatitis C virus was performed. Experimental results showed that DNA samples with concentrations ranging from 10(5) to 10(2)copies/microL can be successfully amplified. Therefore, this new circulating PCR chip may provide a useful platform for genetic identification and molecular diagnosis.

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Section: Resultsmentioning

confidence: 95%

Section: Virus Strain and Pcr Reagentmentioning

confidence: 99%

A micro circulating PCR chip using a suction-type membrane for fluidic transport

Chien

Wang

Hsieh

et al. 2008

Biomed Microdevices

View full text Add to dashboard Cite

show abstract

“…On the day when the work was to be done, samples were brought to room temperature and absorbance values were measured against a blind tube at 260nm and 280nm wavelengths in a spectrophotometer (WPA UV 1101 Biotech Photometer). Then DNA and RNA samples were amplified by Real Time PCR method [6,7,8,9,10].…”

Section: Materıals and Methodsmentioning

confidence: 99%

“…The Real Time PCR technique, which has become increasingly popular in recent times, is a rapid and simple test that allows the quantitation of HBV DNA and HCV RNA [1]. There are many literature related to the subject [6,7,8,9,10].…”

Section: Introductıonmentioning

confidence: 99%

Comparison of the Isolation Methods of Viral Nucleic Acids

Şakrucu¹,

Sıdal

Şanlıdağ

2018

Celal Bayar Üniversitesi Fen Bilimleri Dergisi

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In vitro amplification of the nucleic acids (DNA or RNA) is used in the detection of microbial agents and thus in the diagnosis of infectious diseases, as well as in the diagnoses of oncological and genetic disorders and forensic medicine. The aim of the present study was to compare the isolation methods of the nucleic acids of hepatitis B and C viruses, causative agents of the two significant infections worldwide. Conventional isolation methods were compared with the commercial kits that have been used commonly in recent years, in terms of reliability, cost-effectiveness, contamination risk and duration of the testing time. Five standards for the isolation of the viral nucleic acids of both HBV DNA (Fluorion HBV QNP 2.0) and HCV RNA (Fluorion HCV QNP 2.1) were used. The isolations of the viral nucleic acids of HBV and HCV were done with the conventional methods, phenol-chloroform and guanidine thiocyanate, and the commercial kits Roboscreen and NucleoSpin. The resultant viral nucleic acid load was determined with a spectrophotometer (WPA UV 1101, Biotech Photometer), and their amplification was conducted with Real-Time PCR. The results of the assessments revealed that the highest nucleic acid concentration were obtained with the conventional methods, while they exhibited significant drawbacks such as long duration of the testing time, difficulty in application, and higher contamination risk.

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A novel fluorescence quantification method for polymerase chain reaction system

Cited by 2 publications

References 9 publications

A micro circulating PCR chip using a suction-type membrane for fluidic transport

A micro circulating PCR chip using a suction-type membrane for fluidic transport

Comparison of the Isolation Methods of Viral Nucleic Acids

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