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A polymerase-free paper
LEXINGTON, Mass. & EDINBURGH, U.K.—Quanterix Corp. and DestiNA Genomics Ltd. announced in July that PLOS ONE has published their study detailing a single-probe method for detecting microRNA biomarkers associated with liver toxicity.
The study, entitled “Polymerase-free measurement of microRNA-122 with single base specificity using single molecule arrays: Detection of drug-induced liver injury,” is a proof-of-concept paper showing how the scientists combined Quanterix’s Single Molecule Array Detection technology, or Simoa, and DestiNA’s SMART Nucleobase probe chemistry to develop a test for drug-induced liver toxicity that is at once more accurate and swifter than methods commonly employed today. The study was published jointly by Quanterix, DestiNA Genomics, Edinburgh University and the Pfizer-Universidad de Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENyO).
Drug-induced liver injury (DILI) is caused by prescription and over-the-counter medications or herbal and dietary supplements, and may affect as many as 44,000 people in the United States annually, according to the National Institutes of Health, accounting for 10 percent of all cases of acute hepatitis and over 50 percent of the 2,000 cases of acute liver failure in the United States each year. Those numbers are expected to increase as the use of herbal and dietary supplements rises.
The standard test for DILI had been the alanine transaminase (ALT) enzyme test, but miRNA-122 (miR-122) has recently emerged as a more sensitive and specific biomarker. But testing for miR-122 involved polymerase-chain-reaction (PCR)-based assays and the multiple probes and primers it requires. The new test is a single-probe method for detecting microRNA from human serum using single-molecule arrays, with sequence specificity down to a single base and without the use of amplification by polymerases.
“To optimally manage liver damage as a result of medicines,” Dr. James Dear, reader in clinical pharmacology at the University of Edinburgh and co-author of the paper, said in a media release, “we need to identify biomarkers that can quickly and easily measure drug-induced liver injury. This new method provides an accurate measurement of miR-122 with user-independent sensitivity with a time-to-result that is needed to develop tests that can improve patient safety.”
Using both the single-molecule assay and quantitative PCR, the study measured the miR-122 in the serum of patients who had overdosed on acetaminophen and sustained clinically significant liver injury, as well as a control set from healthy patients. The results show that the sensitivity and specificity of the Simoa assay was sufficient to measure miR-122 in all patients, and showed a clear distinction between healthy controls and those with clinical liver toxicity after drug overdose, as was the case for PCR.
The test employed DestiNA’s aldehyde modified SMART Nucleobases with a unique peptide nucleic acid (PNA) capture probe containing a reactive amine instead of a nucleotide at a specific position in the sequence. The probe was attached to superparamagnetic beads, which were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction.
The beads were then labeled with streptavidin-β-galactosidase (SβG), loaded into an array of femtoliter wells and imaged fluorescently using the Simoa analyzer, which had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mismatch. The combination of the specificity of hybridization between the target and probe sequences, and the single base dynamic chemical incorporation of a label, gave the Simoa assay a high specificity (>3 × 107-fold).
“In terms of applications beyond liver toxicity,” the authors state in the study, “this assay offers a greatly simplified method for the early detection of more specific biomarkers in blood of patients with cancer (so-called liquid biopsies), the early and rapid diagnosis of sepsis, pharmacokinetic measurements of interfering NA therapeutics and the measurement of guide RNA used for gene-editing systems, such as CRISPR/Cas9.”
Hugh Ilyine, DestiNA’s CEO, agrees, saying “We are confident combining the Quanterix Simoa platform with the DestiNA patented reagents will change forever the way RNAs will be analyzed.”
DestiNA and Quanterix have agreed on a timeline for further development of DILI assays based on PCR-free detection of microRNA-122, from patients and test animal serum. Quanterix plans to introduce a new platform which relies on the same core technology as their HD1-Analyzer but will also support various assays for nucleic acid detection. The new system is anticipated to be available to users later this year with a full commercial launch in 2018.