An integrated digital PCR system with high universality and low cost for nucleic acid detection
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Abstract:
Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.Keywords:
Nucleic acid quantitation
Nucleic acid detection
DNA Computing
Droplet digital polymerase chain reaction (ddPCR) is a recently developed method for nucleic acid quantification that allows for more accurate and precise estimation of nucleic acid concentrations. By splitting up a sample into about 14000 droplets using microfluidic technology and subsequently counting the numbers of negative (no nucleic acid was initially present) and positive (nucleic acid was initially present) droplets, the concentration of the nucleic acid can be determined.
Each droplet is classified as positive or negative depending on its fluorescence intensity. Thus, setting a correct fluorescence level threshold is needed to accurately identify the presence or absence of target nucleic acid.
We demonstrate some drawbacks of currently available methods and show how extreme value theory (EVT) can be used to model the maximum fluorescence intensity of the negative droplets using negative control samples (no target nucleic acid present). We show how we subsequently select a final threshold. We discuss some important considerations when applying EVT and how this translates to the ddPCR case.
Nucleic acid detection
Nucleic acid quantitation
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Objective
To explore the differences among three methods of nucleic acid extraction and three kinds of real-time fluorescence quantitative PCR instrument.
Methods
Twenty-five respiratory virus nucleic acid and 25 enterovirus nucleic acid positive samples were with selected at random and nucleic acids were extracted by using three methods (method A, B, and C). The results among different methods were analyzed by randomized block design. 25 respiratory viral nucleic acid positive specimens and enterovirus nucleic acid positive samples were detected by using three kinds of real-time fluorescence quantitative PCR instrument (instrument A, B, and C). The results among different instruments were analyzed by randomized block design.
Results
There was a significant difference among three methods of nucleic acid extraction in results(χ2=42.9162, P 0.05), while method A vs. B, B vs. C were significantly different(Z=7.025, P 0.05), while instrument A vs. B, A vs. C were significantly different(Z=5.70, P<0.001; Z=6.45, P<0.001).
Conclusions
There is difference among different methods and instruments in the test results under the same condition, which call for options in practical work according to need.
Key words:
Nucleic acid extraction; Real-time fluorescence quantitative PCR; Randomized block design
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This chapter contains sections titled: Introduction Detection of nucleic acid targets by nucleic acid probes Functional nucleic acids Fluorescent nucleic acid sensors Colorimetric nucleic acid sensors Electrochemical nucleic acid sensors Piezoelectric nucleic acid sensors Conclusion and perspectives References
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Objective To investigate applied value of automated nucleic acid purification system in clinical detection by real-time fluorescence quantitative PCR. Methods HCV and enteroviruses nucleic acid samples were extracted by nucleic acid extraction apparatus and hand-made method,respectively. The extracted products were detected by fluorescent quantitative PCR. Results The extraction efficiency of automated nucleic acid purification system showed no significant difference between two different magnetic kits(t1=-0.805,P1=005;t4=-1.952,P40.05)The extraction efficiency of automated nucleic acid purification system obviously better than manual method(t=-3.314,P0.01;t=--3.576,P0.01),the extraction efficiency of two different magnetic kits is consistent. extraction of 30 samples used about 2h by manual method. extraction of32 samples used about 1h by automated nucleic acid purification system. Conclusions The extraction efficiency of automated nucleic acid purification system obviously better than hand-made method.NP968 automated nucleic acid purification system is open for different magnetic beads kits. automated nucleic acid purification system is quick,efficient and sample method for extraction and worthy for laboratory applciation.
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The interaction between cyanine Ⅱ with nucleic acids was studied,cyanine Ⅱ fluorescence quenching was obvious in the presence of nucleic acid,and the degree of quenching and the concentration of nucleic acids showed a good linear relationship.
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Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard. Due to these properties, this technique has the potential to not only improve routine quantitative nucleic acid analysis, but also to be used as a reference method for certification of nucleic acid RM. The article focuses on the use and application of both dPCR and RMs for accurate quantification.
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Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.
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