A New Self-Activated Micropumping Mechanism Capable of Continuous-Flow and Real-Time PCR Amplification Inside 3D Spiral Microreactor

A self-activated micropump which is capable of stable velocity transport for a liquid to flow a given distance inside a 3D microchannel has been a dream of microfluidic scientists for a long time. A new self-activated pumping mechanism has been proposed in this paper. It is different from the authors’ previous research which relied on the fluid resistance of a quartz capillary tube or end-blocked gas-permeable silicone or a polydimethylsiloxane (PDMS) wall to automate the flow. In this research, an end-open stretched Teflon tube was utilized for passive transport for the first time. A new fluid transmission mode was adopted with the assistance of a cheaper easily accessible oil mixture to achieve stable continuous flow. Finally, this novel micropump has been applied to real-time continuous-flow polymerase chain reactions (PCRs), with an amplification efficiency similar to that of a commercial PCR cycler instrument.

Download Full-text

A Novel Self-Activated Mechanism for Stable Liquid Transportation Capable of Continuous-Flow and Real-time Microfluidic PCRs

Micromachines ◽

10.3390/mi10060350 ◽

2019 ◽

Vol 10 (6) ◽

pp. 350 ◽

Cited By ~ 1

Author(s):

Di Wu ◽

Bing Shi ◽

Bin Li ◽

Wenming Wu

Keyword(s):

Mathematical Modeling ◽

Real Time ◽

Continuous Flow ◽

Transport Mechanism ◽

The Self ◽

Amplification Efficiency ◽

Passive Transport ◽

Flow Rates ◽

Quartz Capillary ◽

Passive Pumping

The self-activated micropump capable of velocity-stable transport for both single-phased plug and double-phased droplet through long flow distance inside 3D microchannel is one dream of microfluidic scientists. While several types of passive micropumps have been developed based on different actuation mechanisms, until today, it is still one bottleneck to realize such a satisfied self-activated micropump for the stable delivery of both single and double-phased liquid inside long microchannel (e.g., several meters), due to the lack of innovative mechanism in previous methods. To solve this problem, in this article, we propose a new self-activated pumping mechanism. Herein, an end-opened gas-impermeable quartz capillary is utilized for passive transport. Mechanism of this micropump is systemically studied by both the mathematical modeling and the experimental verifications. Based on the flow assays, it totally confirmed a different pumping principle in this paper, as compared with our previous works. The R 2 value of the overall flow rates inside the 3D microchannel is confirmed as high as 0.999, which is much more homogeneous than other passive pumping formats. Finally, this novel micropump is applied to continuous-flow real-time PCRs (both plug-type and microdroplet-type), with the amplification efficiency reaching 91.5% of the commercial PCR cycler instrument.

Download Full-text

Novel real-time polymerase chain reactions for serogroup specific gene detection of Neisseria meningitidis serogroups B, C, W-135 and Y

Journal of Microbiological Methods ◽

10.1016/j.mimet.2013.04.011 ◽

2013 ◽

Vol 94 (2) ◽

pp. 83-85 ◽

Cited By ~ 3

Author(s):

Christine J. Doyle ◽

Amy V. Jennison

Keyword(s):

Neisseria Meningitidis ◽

Real Time ◽

Specific Gene ◽

Gene Detection ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Polymerase Chain

Download Full-text

ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers

Blood ◽

10.1182/blood.v88.5.1852.bloodjournal8851852 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1852-1856 ◽

Cited By ~ 1

Author(s):

C Gassner ◽

A Schmarda ◽

W Nussbaumer ◽

D Schonitzer

Keyword(s):

Pcr Amplification ◽

Abo Blood Groups ◽

Chain Reaction ◽

Specific Primers ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Serological Typing ◽

Modern Molecular Biology ◽

Specific Nucleotide Sequence ◽

Polymerase Chain

Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.

Download Full-text

ABO glycosyltransferase genotyping by polymerase chain reaction using sequence-specific primers

Blood ◽

10.1182/blood.v88.5.1852.1852 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1852-1856 ◽

Cited By ~ 75

Author(s):

C Gassner ◽

A Schmarda ◽

W Nussbaumer ◽

D Schonitzer

Keyword(s):

Pcr Amplification ◽

Abo Blood Groups ◽

Chain Reaction ◽

Specific Primers ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Serological Typing ◽

Modern Molecular Biology ◽

Specific Nucleotide Sequence ◽

Polymerase Chain

Abstract Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.

Download Full-text

A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification

Iranian Journal of Microbiology ◽

10.18502/ijm.v11i2.1085 ◽

2019 ◽

Author(s):

Prabu Siva Sankar ◽

Marimuthu Citartan ◽

Aminah Ahmed Siti ◽

Boris V. Skryabin ◽

Timofey S. Rozhdestvensky ◽

...

Keyword(s):

Dna Polymerase ◽

Expression System ◽

Pcr Amplification ◽

The Other ◽

High Fidelity ◽

Purification Method ◽

Simple Method ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Pfu Dna Polymerase

Background and Objectives: Pfu DNA polymerase is an enzyme that exhibits the lowest error rate in the 3´ to 5´ exonucle- ase (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify Pfu DNA polymerase in a bacterial expression system under a simple purification method. Materials and Methods: Pfu polymerase gene sequence, derived from Pyrocuccus furiosus (Pfu) genomic DNA, was cloned and overexpressed in E. coli BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the Pfu DNA polymerase was heated at 94°C for 5 minutes. Pfu DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable Pfu DNA polymerase was separated from the other debris of the dena- tured proteins by simple centrifugation. Results: The enzymatic activity of the resulting Pfu DNA polymerase was estimated by comparing with the commer- cial Pfu DNA Polymerases. An estimated 50000 units of functional Pfu DNA polymerase was produced from a 400 ml culture. Conclusion: The in-house produced Pfu DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.

Download Full-text

Real-time and multiplex real-time polymerase chain reactions for the detection ofBartonella henselaewithin cat flea,Ctenocephalides felis, samples

Medical and Veterinary Entomology ◽

10.1111/j.1365-2915.2010.00901.x ◽

2010 ◽

Vol 24 (4) ◽

pp. 449-455 ◽

Cited By ~ 8

Author(s):

M. T. ROBINSON ◽

E. R. MORGAN ◽

D. WOODS ◽

S. E. SHAW

Keyword(s):

Real Time ◽

Ctenocephalides Felis ◽

Cat Flea ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Polymerase Chain

Download Full-text

Corrigendum to “Novel real-time polymerase chain reactions for serogroup specific gene detection of Neisseria meningitidis serogroups B, C, W-135 and Y” [J. Microbiol. Meth. 94(2) (2013) 83–85]

Journal of Microbiological Methods ◽

10.1016/j.mimet.2016.08.028 ◽

2016 ◽

Vol 130 ◽

pp. 198

Author(s):

Christine J. Doyle ◽

Amy V. Jennison

Keyword(s):

Neisseria Meningitidis ◽

Real Time ◽

Specific Gene ◽

Gene Detection ◽

Chain Reactions ◽

Polymerase Chain Reactions ◽

Polymerase Chain

Download Full-text

Suitability of Real-Time Quantitative Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for cry9C Detection in Mexican Corn Tortillas: Fate of DNA and Protein After Alkaline Cooking

Journal of AOAC International ◽

10.1093/jaoac/87.3.639 ◽

2004 ◽

Vol 87 (3) ◽

pp. 639-646 ◽

Cited By ~ 12

Author(s):

Maricarmen Quirasco ◽

Bernd Schoel ◽

Javier Plasencia ◽

John Fagan ◽

Amanda Galvez

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Enzyme Linked Immunosorbent Assay ◽

Transgenic Corn ◽

Chain Reactions ◽

Snack Foods ◽

Polymerase Chain Reactions ◽

Corn Products ◽

Polymerase Chain ◽

Corn Kernels

Abstract Alkaline-cooked corn, called nixtamal, is the basis for many traditional corn products such as tortillas, chips, and taco shells that are used widely in Mexico and Central America and in the preparation of snack foods that are consumed globally. To assess the effects of alkaline and thermal treatments on the detectability of DNA and protein for the presence of genetically modified sequences, various nixtamalized products were prepared from blends of conventional white corn containing 0.1, 1.0, and 10% transgenic corn (event CBH 351, StarLink™). Real-time quantitative polymerase chain reactions (RTQ–PCR) and immunoassays were used to determine the cry9C gene and protein, respectively, in unprocessed corn kernels, freshly prepared alkaline-cooked and ground corn (masa), masa flour, tortillas prepared from masa by heat treatment, chips prepared from damp masa dough by deep frying, and from tortillas processed at high (200°C) and low temperatures (70°C). In spite of progressive degradation of genomic DNA during processing, RTQ–PCR genetic analysis allowed detection and quantification of the cry9C gene in all products prepared from 10, 1, and 0.1% StarLink corn, except deep-fried chips containing 0.1% StarLink. Enzyme-linked immunosorbent assays readily detected <1ppm cry9C protein in all blends of unprocessed corn (10, 1, and 0.1% StarLink) as well as in nonfried tortilla and masa products. This technique was not suitable for thermally treated nixtamalized products containing <1% transgenic corn.

Download Full-text