Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers

Abstract

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-01878-z) contains supplementary material, which is available to authorized users.

Physical and chemical template-blocking strategies in the exponential amplification reaction of circulating microRNAs

“…With the highly significant clinical relevance and disease implications entailed in these miRNAs, an isothermal assay that can rapidly quantify their abundance in biological matrices with simple procedures will be highly beneficial for validating their roles as diagnostic markers in different diseases. However, while many isothermal methods have been reported for detection of miR-16 and miR-21 [59][60][61][62][63], relatively few assays have been developed so far for circulating miR-122 [64][65][66]. The Bst homologs, Bst 2.0, Bst 2.0 WarmStart, and Bst 3.0, are in silico designs based on Bst large fragment but with improved processivity and thermostability.…”

Fluorescence calibration standards made from broadband emitters encapsulated in polymer beads for fluorescence microscopy and flow cytometry

“…The suitability of the dye-based spectral fluorescence standards from the BAM Kit for the determination of the emission correction curve s ( λ em ) −1 motivated us to exploit these dyes also for the design of candidate bead-based spectral standards. Such calibration beads are much closer to the many rapidly evolving (bio)analytical applications of particulate labels and bead-based platforms, e.g., in suspension microparticle assays [ 34 – 39 ] as well as to eukaryotic and bacteria cells typically measured with fluorescence microscopy techniques than dye solutions. Unlike beads, dye solutions are also not suited for the calibration and IPV of FCM instruments that present another goal of our research focused on the development of calibration and standardization concepts for optical-spectroscopic techniques.…”

Ultrasound-Enhanced Chemiluminescence for Bioimaging

“…tumor markers (a-fetoprotein), thyroid stimulating hormone (TSH) and immunoglobulins (Ma et al, 2017;Min et al, 2018;Chen et al, 2019). Acridinium ester is also able to successfully label strands of DNA to produce DNA probes to emit CL (Komori et al, 2019). In a study, wild-type p53 was immobilized on the surface of gold-functionalized magnetic nanoparticles.…”