A Rapid Digital PCR System with a Pressurized Thermal Cycler

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.

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Rapid Real-Time Fluorescent PCR Gene Dosage Test for the Diagnosis of DNA Duplications and Deletions

Clinical Chemistry ◽

10.1093/clinchem/46.10.1574 ◽

2000 ◽

Vol 46 (10) ◽

pp. 1574-1582 ◽

Cited By ~ 42

Author(s):

Clara Ruiz-Ponte ◽

Lourdes Loidi ◽

Ana Vega ◽

Angel Carracedo ◽

Francisco Barros

Keyword(s):

Real Time ◽

Capillary Tube ◽

Gene Dosage ◽

Melting Curve ◽

Gene Copy Number ◽

Gene Copy ◽

Target Sequence ◽

Hereditary Neuropathy ◽

Target Dna ◽

Fluorescent Pcr

Abstract Background: Current methods to determine gene dosage are time-consuming and labor-intensive. We describe a new and rapid method to assess gene copy number for identification of DNA duplications or deletions occurring in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively. Methods: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed. Results: Area ratios were ∼0.6, 1.0, and 2.0 for HNPP, control, and CMT1A samples, respectively. The results agreed with those obtained by Southern blotting and microsatellite analysis in the same samples. Conclusions: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.

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Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA

Bioscience Biotechnology and Biochemistry ◽

10.1080/09168451.2016.1196576 ◽

2016 ◽

Vol 80 (11) ◽

pp. 2159-2164 ◽

Cited By ~ 17

Author(s):

Hui Tang ◽

Qingchun Cai ◽

Hu Li ◽

Peng Hu

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Real Time ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Hepatitis B Virus Dna ◽

B Virus

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Could Droplet Digital PCR Be Used Instead of Real-Time PCR for Quantitative Detection of the Hepatitis B Virus Genome in Plasma?: TABLE 1

Journal of Clinical Microbiology ◽

10.1128/jcm.01306-14 ◽

2014 ◽

Vol 52 (9) ◽

pp. 3497-3498 ◽

Cited By ~ 14

Author(s):

Laure Boizeau ◽

Syria Laperche ◽

Nathalie Désiré ◽

Catherine Jourdain ◽

Vincent Thibault ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Real Time ◽

Real Time Pcr ◽

Digital Pcr ◽

Virus Genome ◽

Droplet Digital Pcr ◽

Quantitative Detection ◽

B Virus

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Faculty Opinions recommendation of Comparison of real-time PCR assays for monitoring serum hepatitis B virus DNA levels during antiviral therapy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1040569.489566 ◽

2006 ◽

Author(s):

David Persing

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Therapy ◽

Real Time ◽

Real Time Pcr ◽

Serum Hepatitis ◽

Hepatitis B Virus Dna ◽

B Virus ◽

Pcr Assays

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Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

European Food Research and Technology ◽

10.1007/s00217-021-03786-y ◽

2021 ◽

Author(s):

Christian Schulze ◽

Anne-Catrin Geuthner ◽

Dietrich Mäde

Keyword(s):

Durum Wheat ◽

Real Time ◽

Bread Wheat ◽

Common Wheat ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Food Fraud ◽

Single Genome ◽

Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

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Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

Animals ◽

10.3390/ani11051270 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1270

Author(s):

Anna Cutarelli ◽

Andrea Fulgione ◽

Pasquale Fraulo ◽

Francesco Paolo Serpe ◽

Pasquale Gallo ◽

...

Keyword(s):

Digital Pcr ◽

Buffalo Milk ◽

Cow Milk ◽

Mozzarella Cheese ◽

Chain Reaction ◽

New Methods ◽

Protected Designation Of Origin ◽

Commission Regulation ◽

Polymerase Chain ◽

Digital Polymerase Chain Reaction

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

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