Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications

Cao, Lei; Cui, Xingye; Hu, Jie; Li, Zedong; Choi, Jane Ru; Yang, Qingzhen; Lin, Min; Hui, Li Ying; Xu, Feng

doi:10.1016/j.bios.2016.09.082

Cited by 230 publications

(151 citation statements)

References 213 publications

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“…Considering the cost of ddPCR is estimated to be five times that of TaqMan qPCR[33] while qPCR allows the processing of more samples per run, we agree with the suggestion that ddPCR is optimal for calibrating targets for subsequent use on other platforms such as qPCR[22,23,35]. …”

Section: Discussionsupporting

confidence: 84%

“…One of the main advantages of ddPCR is that it provides absolute quantification of the target DNA in copy number to a point where it has been proposed for the construction of calibration curves[22,23,35]. Direct comparison of the results obtained with the two techniques revealed strong correlations between ddPCR and qPCR measurements.…”

Section: Discussionmentioning

confidence: 99%

“…Besides the obvious interest in the detection of rare mutations of various kinds, ddPCR technology displays a number of additional advantages for general nucleic acid quantification[23,24] such as the capability to obtain absolute quantification without the need of standard dilution curves and reaction efficiency determination[22,23]. Another potential advantage of ddPCR technology, being an end-point approach, is the reaction tolerance to PCR inhibitors[22], an important advantage when using clinical samples[25].…”

Section: Introductionmentioning

confidence: 99%

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Droplet digital PCR for quantification ofITGA6in a stool mRNA assay for the detection of colorectal cancers

Herring

Kanaoka

Tremblay

et al. 2017

WJG

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AIMTo investigate the use of droplet digital polymerase chain reaction (ddPCR) for detecting host mRNA markers in stools as a non-invasive test for colorectal cancer screening.METHODSddPCR and quantitative PCR were compared side by side for their performance in the detection of ITGA6 and ITGA6A transcripts in stool samples obtained from patients with various types of colorectal lesions (advanced adenomas and stage II-IV colorectal cancers) and control (patients displaying no pathological findings) using duplex TaqMan reactions for both methods. ITGA6 and ITGA6A were chosen for this proof-of-concept study based on their relative medium and low abundance in stool samples, respectively, as established in a previous study.RESULTSWe found that the ddPCR and qPCR methods performed equally well in this TaqMan duplex assay for the detection of ITGA6 and ITGA6A transcripts in stools of patients with colorectal lesions. For ITGA6, receiver operating characteristic (ROC) curve analysis showed comparable areas under the curve of 0.91 (P < 0.0001) and 0.89-0.90 (P < 0.0001) for the prediction of advanced adenomas and colorectal cancers, respectively. ITGA6A, which was detected at very low levels in control patients, was found to be significantly elevated (over 40 times) in stage II and III colorectal cancers (P < 0.0002). Comparison of the two sets of data revealed a strong correlation of the copy numbers obtained by ddPCR and qPCR for both ITGA6 and ITGA6A.CONCLUSIONWe found that ITGA6 and ITGA6A detection in stools of patients with colorectal cancers with ddPCR is comparable to that of qPCR using TaqMan assays.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Droplet digital PCR for quantification ofITGA6in a stool mRNA assay for the detection of colorectal cancers

Herring

Kanaoka

Tremblay

et al. 2017

WJG

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“…An initial denaturation of DNA template at 95°C for 2 min followed by a denaturation step for 45 sec at 94°C, an annealing step for 45 sec at optimum temperature (60°C for Bs fragment and 67°C for the Ap fragment) and an extension reaction at 72°C for 1 min. After last PCR cycle, a final extension step for 2 min at 72 °C was added [37]. The amplified products were then digested with BsmI, ApaI and TaqI restriction endonucleases overnight at 37°C, electrophoresed on 2% horizontal agarose gel, stained with 0.5 mg/mL of ethidium bromide and visualized under UVBillumination using the E-Gel Precast Agarose Electrophoresis System (Invitrogen Life Technologies, PA4 9RF Paisley, UK) [38].…”

Section: Detection Of Vdr Gene Polymorphismsmentioning

confidence: 99%

The Significance of Vitamin D Receptor Gene Polymorphisms for Susceptibility to Hepatocellular Carcinoma in Subjects Infected with Hepatitis C Virus

Mohammed¹

2017

GHOA

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Background: Vitamin D has emerging roles in fibrogenesis, cell cycle arrest, immune modulation, and tumorigenesis. Several single nucleotide polymorphisms (SNPs) in vitamin D receptor (VDR) gene are associated with tumorigenesis in various organs.Objective: to investigate the association between the VDR gene polymorphisms and Hepatocellular carcinoma (HCC) risk and severity in Egyptian patients with chronic hepatitis C (CHC). Methods:Five hundred thirty outpatients with chronic hepatitis C virus (HCV) infection were initially enrolled, of which 180 patients with CHC, 180 patients with liver cirrhosis and 170 patients with HCC. Another 170 age-and sex-matched healthy controls were enrolled. Genotyping of VDR gene at BsmI, ApaI and TaqI loci was performed. Evaluation of clinicopathological features of HCC was done. Data were analyzed using SPSS software (Version 17.0). Results Conclusion:The present study provided significant associations of VDR gene SNPs (ApaI CC genotype and bAt[CCA]-haplotype) with HCC development and disease severity in HCV-infected Egyptian patients. Thus, the determination of these VDR genetic variants in HCV patients could help identification of patients at-risk of HCC development.

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“…As the essential part of dPCR, the distribution of the template is mainly based on microwell chip, water-in-oil droplet and microfluidic techniques [2]. Among these methods, the water-in-oil droplet technique is the most frequently used, and the related droplet digital PCR (ddPCR) has been commercialized.…”

Section: Digital Pcrmentioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications

Cited by 230 publications

References 213 publications

Droplet digital PCR for quantification ofITGA6in a stool mRNA assay for the detection of colorectal cancers

Droplet digital PCR for quantification ofITGA6in a stool mRNA assay for the detection of colorectal cancers

The Significance of Vitamin D Receptor Gene Polymorphisms for Susceptibility to Hepatocellular Carcinoma in Subjects Infected with Hepatitis C Virus

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

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