Sensitive detection of <i>Porcine circovirus-2</i> by droplet digital polymerase chain reaction

Zhao, Shan; Lin, Hua; Chen, Shijie; Yang, Miao; Yan, Qigui; Wen, Caifang; Zhang, Hao; Yan, Yubao; Sun, Yingxun; Hu, Juan; Chen, Zhenrong; Xi, Lixin

doi:10.1177/1040638715608358

Cited by 31 publications

(28 citation statements)

References 16 publications

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“…Results are expressed as target copies per microliter of reaction [ 31 ]. In comparison with qPCR, the ddPCR technique has some favorable features, among which: 1) it performs absolute quantification based on the principles of sample partitioning and Poisson statistics, thus overcoming the normalization and calibrator issues [ 32 ]; 2) it has shown increased precision [ 33 ] and sensitivity in detecting low target copies [ 34 ]; 3) it is relatively insensitive to potential PCR inhibitors [ 35 – 37 ]; 4) it directly provides the result of the analysis expressed as number of copies of target per microliter of reaction (with confidence intervals) [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

et al. 2016

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BackgroundSelected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum.ResultsIn the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001).To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample.ConclusionsAnalyses of serum miRNAs performed with qPCR and ddPCR were largely concordant. Both qPCR and ddPCR can reliably be used to quantify circulating miRNAs, however, ddPCR revealed similar or greater precision and higher throughput of analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0292-7) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

et al. 2016

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“…A number of studies on ddPCR show high sensitivity of the method and high precision for absolute quantification of DNA targets, for example in cancer [5–8], hepatitis B virus [9, 10], cytomegalovirus[11, 12], Chlamydia trachomatis [13, 14], Babesia microti , and Babesia duncani [15]. Comparison of ddPCR assays with real-time PCR assays showed that ddPCR assays had a higher sensitivity than real-time PCR assays [16, 17], but this was not confirmed in some other studies [11, 18]. Real-time PCR assays are widely used for detection and relative quantification of Plasmodium parasites.…”

Section: Introductionmentioning

confidence: 99%

Four human Plasmodium species quantification using droplet digital PCR

et al. 2017

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Droplet digital polymerase chain reaction (ddPCR) is a partial PCR based on water-oil emulsion droplet technology. It is a highly sensitive method for detecting and delineating minor alleles from complex backgrounds and provides absolute quantification of DNA targets. The ddPCR technology has been applied for detection of many pathogens. Here the sensitive assay utilizing ddPCR for detection and quantification of Plasmodium species was investigated. The assay was developed for two levels of detection, genus specific for all Plasmodium species and for specific Plasmodium species detection. The ddPCR assay was developed based on primers and probes specific to the Plasmodium genus 18S rRNA gene. Using ddPCR for ultra-sensitive P. falciparum assessment, the lower level of detection from concentrated DNA obtained from a high volume (1 mL) blood sample was 11 parasites/mL. For species identification, in particular for samples with mixed infections, a duplex reaction was developed for detection and quantification P. falciparum/ P. vivax and P. malariae/ P. ovale. Amplification of each Plasmodium species in the duplex reaction showed equal sensitivity to singleplex single species detection. The duplex ddPCR assay had higher sensitivity to identify minor species in 32 subpatent parasitaemia samples from Cambodia, and performed better than real-time PCR. The ddPCR assay shows high sensitivity to assess very low parasitaemia of all human Plasmodium species. This provides a useful research tool for studying the role of the asymptomatic parasite reservoir for transmission in regions aiming for malaria elimination.

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“…In our present study, we used 3D digital PCR to accurately quantify the low levels of miRNA for validation in a large number of samples. Digital PCR, which is a third generation of PCR, has increased precision and sensitivity for detecting low amounts of target copies when compared to conventional quantitative real-time PCR (29,30). As such, it can be used for the detection and quantification of low levels of pathogens, rare genetic sequences, and cancer-related miRNAs (31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

miR‑3940‑5p/miR‑8069 ratio in urine exosomes is a novel diagnostic biomarker for pancreatic ductal adenocarcinoma

et al. 2020

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Despite the development of several therapeutic options, the prognosis of pancreatic cancer remains poor. One reason for this is the difficulty of diagnosing the disease at an early stage. For example, carbohydrate antigen (CA) 19-9, which is the most widely used biomarker for pancreatic cancer, cannot be used to detect the disease at early stages. Some studies have attempted to find novel biomarkers for pancreatic cancer. The aim of the present study was to find a novel diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC) in urine exosomes. Exosomes were isolated from urine and serum samples of patients with PDAC and control subjects, or culture media of cancer cell lines. MicroRNAs (miRNAs) were purified from exosomes. Novel biomarker candidates for PDCA were identisfied from urine exosome miRNA using expression profiling, and validated in a larger number of samples using 3D digital PCR. The results of a preliminary analysis of nine PDAC and seven control subjects revealed that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in the patients with PDAC. Experiments using cultured cancer cell lines revealed that the elevation of the miR-3940-5p/miR-8069 ratio was specific for PDAC. Furthermore, the elevation of the miR-3940-5p/miR-8069 ratio in exosomes tended to be higher in the urine than in the serum of patients with PDAC. Validation experiments on 43 PDAC, 12 chronic pancreatitis and 25 control subjects demonstrated that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in PDAC at a relatively early stage of the disease. When this ratio was used in combination with CA19-9 for the diagnosis of PDAC, the sensitivity and positive predictive value improved to 93.0 and 78.4%, respectively, when either of them was positive. Additionally, the positive predictive value reached 100% when both were positive. The negative predictive value also improved to 89.7% when both were negative. The miR-3940-5p/miR-8069 ratio in urine exosomes may be useful as a tool for the diagnosis of PDAC, particularly when used in combination with CA19-9.

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Sensitive detection of Porcine circovirus-2 by droplet digital polymerase chain reaction

Cited by 31 publications

References 16 publications

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

Four human Plasmodium species quantification using droplet digital PCR

miR‑3940‑5p/miR‑8069 ratio in urine exosomes is a novel diagnostic biomarker for pancreatic ductal adenocarcinoma

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