Dataset Information

Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

ABSTRACT: Novel non-invasive biomarkers for gastric cancer (GC) are needed, because the present diagnostic methods for GC are either invasive or insensitive and non-specific in clinic. The presence of stable circulating microRNAs (miRNAs) in plasma suggested a promising role as GC biomarkers.Based on the quantitative droplet digital PCR (ddPCR), four miRNAs (miR-21, miR-93, miR-106a and miR-106b) related to the presence of GC were identified in plasma from a training cohort of 147 participants and a validation cohort of 28 participants.All circulating miRNA levels were significantly higher in the plasma of GC patients compared to healthy controls (P?

SUBMITTER: Zhao G

PROVIDER: S-EPMC6013872 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

Zhao Gaoping G Jiang Tao T Liu Yanzhuo Y Huai Guoli G Lan Chunbin C Li Guiquan G Jia Guiqing G Wang Kang K Yang Maozhu M

BMC cancer 20180622 1

<h4>Background</h4>Novel non-invasive biomarkers for gastric cancer (GC) are needed, because the present diagnostic methods for GC are either invasive or insensitive and non-specific in clinic. The presence of stable circulating microRNAs (miRNAs) in plasma suggested a promising role as GC biomarkers.<h4>Methods</h4>Based on the quantitative droplet digital PCR (ddPCR), four miRNAs (miR-21, miR-93, miR-106a and miR-106b) related to the presence of GC were identified in plasma from a training coh ...[more]

PMID: 29929476

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Project description:BackgroundDroplet digital PCR (ddPCR) has emerged as a promising tool of pathogen detection in bloodstream infections (BSIs) in critical care medicine. However, different ddPCR platforms have variable sensitivity and specificity for diverse microorganisms at various infection sites. There is still a lack of prospective clinical studies aimed at validating and interpreting the discrepant ddPCR results for diagnosing BSI in intensive care unit (ICU) practice.MethodsA prospective diagnostic study of multiplex ddPCR panels was conducted in a general ICU from May 21, 2021, to December 22, 2021. Paired blood cultures (BCs) and ddPCRs (2.5 h) were obtained synchronously to detect the 12 most common BSI pathogens and three antimicrobial resistance (AMR) genes. Firstly, ddPCR performance was compared to definite BSI. Secondly, clinical validation of ddPCR was compared to composite clinical diagnosis. Sensitivity, specificity, and positive and negative predictive values were calculated. Thirdly, the positive rate of AMR genes and related analysis was presented.ResultsA total of 438 episodes of suspected BSIs occurring in 150 critical patients were enrolled. BC and ddPCR were positive for targeted bacteria in 40 (9.1%) and 180 (41.1%) cases, respectively. There were 280 concordant and 158 discordant. In comparison with BCs, the sensitivity of ddPCR ranged from 58.8 to 86.7% with an aggregate of 72.5% in different species, with corresponding specificity ranging from 73.5 to 92.2% with an aggregate of 63.1%. Furthermore, the rate of ddPCR+/BC- results was 33.6% (147/438) with 87.1% (128 of 147) cases was associated with probable (n = 108) or possible (n = 20) BSIs. When clinically diagnosed BSI was used as true positive, the final sensitivity and specificity of ddPCR increased to 84.9% and 92.5%, respectively. In addition, 40 blaKPC, 3blaNDM, and 38 mecA genes were detected, among which 90.5% were definitely positive for blaKPC. Further, 65.8% specimens were predicted to be mecA-positive in Staphylococcus sp. according to all microbiological analysis.ConclusionsThe multiplexed ddPCR is a flexible and universal platform, which can be used as an add-on complementary to conventional BC. When combined with clinical infection evidence, ddPCR shows potential advantages for rapidly diagnosing suspected BSIs and AMR genes in ICU practice.

Project description:Droplet digital PCR (ddPCR) is being advocated as a reference method to measure rare genomic targets. It has consistently been proven to be more sensitive and direct at discerning copy numbers of DNA than other quantitative methods. However, one of the largest obstacles to measuring microRNA (miRNA) using ddPCR is that reverse transcription efficiency depends upon the target, meaning small RNA nucleotide composition directly effects primer specificity in a manner that prevents traditional quantitation optimization strategies. Additionally, the use of reagents that are optimized for miRNA measurements using quantitative real-time PCR (qRT-PCR) appear to either cause false positive or false negative detection of certain targets when used with traditional ddPCR quantification methods. False readings are often related to using inadequate enzymes, primers and probes. Given that two-step miRNA quantification using ddPCR relies solely on reverse transcription and uses proprietary reagents previously optimized only for qRT-PCR, these barriers are substantial. Therefore, here we outline essential controls, optimization techniques, and an efficacy model to improve the quality of ddPCR miRNA measurements. We have applied two-step principles used for miRNA qRT-PCR measurements and leveraged the use of synthetic miRNA targets to evaluate ddPCR following cDNA synthesis with four different commercial kits. We have identified inefficiencies and limitations as well as proposed ways to circumvent identified obstacles. Lastly, we show that we can apply these criteria to a model system to confidently quantify miRNA copy number. Our measurement technique is a novel way to quantify specific miRNA copy number in a single sample, without using standard curves for individual experiments. Our methodology can be used for validation and control measurements, as well as a diagnostic technique that allows scientists, technicians, clinicians, and regulators to base miRNA measures on a single unit of measurement rather than a ratio of values.

Dataset Information

Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

Publications

Droplet digital PCR-based circulating microRNA detection serve as a promising diagnostic method for gastric cancer.

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