DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

BACKGROUND: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. RESULTS: We herein describe a UDG-REtarded CRISPR Amplification assay, termed 'URECA'. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10-4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SIGNIFICANCE: URECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe.

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0-10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

Complete genome sequence of a new putative member of the genus Pelarspovirus that infects Quercus aliena Blume in South Korea.

The complete nucleotide sequence of a newly discovered virus infecting Quercus aliena Blume, tentatively named "quercus leafroll virus" (QLRV), was determined through high-throughput and Sanger sequencing. The sequence comprises 3,940 nucleotides, has five open reading frames, and has a typical pelarspovirus genome organization, with neither 3' polyadenylation nor a 5' cap. The proteins encoded by QLRV share 17.9 to 44.2% amino acid sequence identity with known pelarspovirus proteins. The highest amino acid sequence identity values for the RNA-dependent RNA polymerase (RdRp) and coat protein were 67.5% and 55.2%, respectively, which are below the current thresholds for pelarspovirus species demarcation. On the basis of these results, we propose classifying QLRV as a new member of the genus Pelarspovirus, family Tombusviridae.

First report of Kalanchoe Latent Virus naturally infecting common bean (Phaseolus vulgaris L.) in South Korea.

The common bean (Phaseolus vulgaris; family: Fabaceae) is an economically and nutritionally important food crop worldwide (Ganesan et al. 2017). In 2021, several plants collected from different provinces in South Korea had symptoms of viral infections (e.g., mild yellow-greenish speckling, stunting, crinkling, and deformed leaves). To identify the causal pathogens, total RNA was isolated from pooled leaf tissues from all samples (n = 29) for paired-end high-throughput sequencing (HTS). The cDNA library was constructed after eliminating ribosomal RNA using the TruSeq RNA Sample Prep Kit and then sequenced using the Illumina NovaSeq 6000 platform (Macrogen, Korea). The 297,868,156 paired-end clean reads (150 nt) were de novo assembled using Trinity with default parameters. BLASTx was used for the contig analysis, which revealed the pooled samples were infected with several plant viruses (e.g., turnip mosaic virus, zucchini yellow mosaic virus, cucumber mosaic virus, lily mottle virus). Notably, the assembled contigs included a single viral contig (8,472 nt) comprising the nearly complete KLV genome (HTS mean coverage: 39.46%). Kalanchoe latent virus (KLV; genus: Carlavirus; family: Betaflexiviridae) has been detected in Kalanchoë blossfeldiana (Hearon 1982), Chenopodium quinoa (Dinesen et al. 2009), and Graptopetalum paraguayense (Sorrentino et al. 2017). The sequence was most similar (96.28% nucleotide identity; 99% query coverage) to KLV isolate DSMZ PV-0290 (GenBank: OP525283) from Denmark. The contig sequence was validated via reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from the 29 individually stored samples and nine primer sets specific for the KLV contig. All nine contig-specific overlapping fragments were amplified from only a P. vulgaris plant with mild yellowing mosaic symptoms collected on July 6, 2021, in Jeongseon County, South Korea. Additionally, 5' and 3' rapid amplification of cDNA ends (RACE)-specific primers were designed for the KLV contig sequence to determine the terminal ends of the genome of the South Korean KLV isolate using the 5'/3' RACE System (Invitrogen, Carlsbad, CA, USA). All of the amplified and overlapping fragments were cloned into the RBC T&A Cloning Vector (RBC Bioscience, Taipei, Taiwan) and sequenced using the Sanger method. The obtained full-length genomic sequence of the KLV isolate (KLV-SK22) was 8,517 nt long and was deposited in GenBank OQ718816. According to the BLASTn analysis, KLV-SK22 was highly similar (96.30% sequence identity; 100% query coverage) to the DSMZ PV-0290 isolate. Phylogenetic trees constructed on the basis of coat protein and RNA-dependent RNA polymerase amino acid sequences revealed that KLV-SK22 is closely related to the DSMZ PV-0290 and PV-0290B isolates from Denmark, respectively. At the genome and gene levels, the individual sequence identities between the carlaviruses and other KLV isolates were 96.29% to 100% (Adams et al. 2004). Additionally, an RT-PCR analysis using detection primers specific for KLV-SK22 did not detect KLV in 15 samples (P. vulgaris = 3, Glycine max = 8, Pueraria montana = 2, Trifolium repens = 1, and Vigna angularis = 1) randomly collected from different regions in South Korea. Based on these results, KLV infection may not be widespread at this time in South Korea. To the best of our knowledge, this is the first report of KLV in P. vulgaris in South Korea or elsewhere. Our findings will aid future research on the epidemiology and long-term management of KLV-related diseases.

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5'-end of a hairpin probe (HP), forming a blunt end. When the 3'-end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3'-end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

Background-filtered telomerase activity assay with cyclic DNA cleavage amplification.

Overexpression of telomerase incites the abnormal proliferation of cancer cells. Thus, it has been regarded as a cancer biomarker and a potential therapeutic target. Existing assays suggest a promising sensing scheme to detect telomerase activity. However, they are complicated in terms of assay preparation and implementation. We herein report a Quenching-Exempt invader Signal Amplification Test, termed 'QUEST'. The assay leverages on a high turnover, specific cleaving enzyme, flap endonuclease I (FEN1), and graphene oxide (GO) for background (BG) filtering. In response to the target, FEN1 significantly boosts the signal with invader signal amplification. To distinguish the target signal, GO filters out the BG. It captures residual reporter invader probes (RP) to quench undesired signals. QUEST is straightforward without any pre-preparatory steps and washing/separation. Its probe design is simple and cost-effective. With QUEST, we investigated telomerase activities in various cell lines. Notably, we discriminated cancer cell lines from normal cell lines. In addition, a candidate inhibitor for telomerase was screened, which showed the promising potential of QUEST in real applications.

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria's activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

Complete genome sequence of cnidium virus 2, a novel cytorhabdovirus isolated from Cnidium officinale in South Korea.

High-throughput sequencing identified a cytorhabdovirus, tentatively named "cnidium virus 2" (CnV2), in Cnidium officinale, and Sanger sequencing confirmed the genome sequence. CnV2 is 13,527 nucleotides in length and contains seven open reading frames in the order 3'-N-P-3-4-M-G-L-5', separated by intergenic regions. The full-length nucleotide sequence of CnV2 shares 19.4-53.8% identity with other known cytorhabdovirus genome sequences. The N, P, P3, M, G, and L proteins share 15.8-66.7%, 11-64.3%, 11.1-80.5%, 10.8-75.3%, 12.3-72.1%, and 20-72.7% amino acid sequence identity, respectively, with the cognate deduced protein sequences from known cytorhabdoviruses. CnV2 is related to other members of the genus Cytorhabdovirus, with sambucus virus 1 being the closest relative. Thus, CnV2 should be classified as a new member in the genus Cytorhabdovirus of the family Rhabdoviridae.

Complete genome sequence of daphne virus 1, a novel cytorhabdovirus infecting Daphne odora.

A novel cytorhabdovirus was identified in Daphne odora in South Korea using high-throughput sequencing. The virus, tentatively named "daphne virus 1" (DV1), has a full-length genome sequence of 13,206 nucleotides with a genome organization comparable to that of unsegmented plant rhabdoviruses and contains seven antisense putative genes in the order 3'-leader-N-P'-P-P3-M-G-L-5'-trailer. The coding region of the genome is flanked by a 3' leader and a 5' trailer sequence, 261 and 151 nucleotides long, respectively. The DV1 genome shares 33.74%-57.44% nucleotide sequence identity with other cytorhabdoviruses. The DV1-encoded proteins share the highest amino acid sequence identity with homologues from Asclepias syriaca virus 1. Phylogenetic analysis showed that DV1 clustered with representative cytorhabdoviruses. We propose classifying DV1 in a new species within the genus Cytorhabdovirus, family Rhabdoviridae.

Complete genome sequence of a novel member of the genus Polerovirus from Cnidium officinale in South Korea.

The complete genome sequence of a novel virus found infecting Cnidium officinale, which we have named "cnidium polerovirus 1" (CnPV1), is 6,090 nucleotides in length, similar to those of other poleroviruses. Seven open reading frames (ORF0-5 and ORF3a) were predicted in this genome. CnPV1 shares 32.4%-38.9% full-length nucleotide sequence identity with other known polerovirus genome sequences. The putative P0, P1-2, P3-5, P3, and P4 proteins share 11.3%-19.5%, 37.1%-49.8%, 26.7%-39.5%, 40.8%-49.7%, and 40.8%-49.7% amino acid sequence identity, respectively, with homologous inferred protein sequences from known poleroviruses. Phylogenetic analysis of P1-2 and P3 sequences places CnPV1 with other members of the genus Polerovirus, indicating that it should be classified in a new distinct species.

The complete genome sequence of Stellaria aquatica virus A, a new member of the genus Alphacarmovirus, family Tombusviridae.

A new member of the genus Alphacarmovirus was detected in Stellaria aquatica using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named "Stellaria aquatica virus A" (StAV-A), comprises 4,017 nucleotides with five predicted open reading frames (ORFs) and has a typical alphacarmovirus genome organization. Pairwise comparison of StAV-A with selected members of family Tombusviridae showed 44-58%, 32-64%, and 19-49% sequence identity for the overall nucleotide sequence, polymerase, and coat protein, respectively. Phylogenetic analysis of polymerase sequences places StAV-A alongside other members of the genus Alphacarmovirus in the family Tombusviridae.

CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter.

Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2',3'-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.

Washing-Free and Label-Free Onsite Assay for Inorganic Pyrophosphatase Activity Using a Personal Glucose Meter.

In this study, we demonstrated a personal glucose meter-based method for washing-free and label-free inorganic pyrophosphatase (PPase) detection, which relies on the cascade enzymatic reaction (CER) promoted by hexokinase and pyruvate kinase. In principle, the absence of target PPase enables adenosine triphosphate sulfurylase to catalyze the conversion of pyrophosphate (PPi) to ATP, a substrate of CER, which results in the significant reduction of glucose levels by the effective CER process. In contrast, the PPi cleavage activity works in the presence of target PPase by decomposing PPi to orthophosphate (Pi). Therefore, the CER process cannot be effectively executed, leading to the maintenance of the initial high glucose level that may be measured by a portable personal glucose meter. Based on this novel strategy, a quantitative evaluation of the PPase activity may be achieved in a dynamic linear range of 1.5-25 mU/mL with a detection limit of 1.18 mU/mL. Compared with the previous PPase detection methods, this method eliminates the demand for expensive and bulky analysis equipment as well as a complex washing step. More importantly, the diagnostic capability of this method was also successfully verified by reliably detecting PPase present in an undiluted human serum sample with an excellent recovery ratio of 100 ± 2%.

Immunoglobulin E Detection Method Based on Cascade Enzymatic Reaction Utilizing Portable Personal Glucose Meter.

We herein describe a cascade enzymatic reaction (CER)-based IgE detection method utilizing a personal glucose meter (PGM), which relies on alkaline phosphatase (ALP) activity that regulates the amount of adenosine triphosphate (ATP). The amount of sandwich assay complex is determined according to the presence or absence of the target IgE. Additionally, the ALP in the sandwich assay catalyzes the dephosphorylation of ATP, a substrate of CER, which results in the changes in glucose level. By employing this principle, IgE was reliably detected at a concentration as low as ca. 29.6 ng/mL with high specificity toward various proteins. Importantly, the limit of detection (LOD) of this portable PGM-based approach was comparable to currently commercialized ELISA kit without expensive and bulky analysis equipment as well as complexed washing step. Finally, the diagnostic capability of this method was also successfully verified by reliably detecting IgE present in a real human serum sample with an excellent recovery ratio within 100 ± 6%.

Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing a nicking and extension chain reaction system.

Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA promoter-containing double-stranded DNA (T7DNA), and thus the final amplification efficiency is not sufficiently high enough to achieve ultrasensitive detection. We herein ingeniously integrate a nicking and extension chain reaction system into the NASBA to establish an ultrasensitive version of NASBA, termed Nicking and Extension chain reaction System-Based Amplification (NESBA). By employing a NESBA primer set designed to contain an additional nicking site at the 5' end of a NASBA primer set, the T7DNA is exponentially amplified through continuously repeated nicking and extension chain reaction by the combined activities of nicking endonuclease (NE) and reverse transcriptase (RT). As a consequence, a much larger number of RNA amplicons would be produced through the transcription of the amplified T7DNA, greatly enhancing the final fluorescence signal from the molecular beacon (MB) probe binding to the RNA amplicon. Based on this unique design principle, we successfully identified the target respiratory syncytial virus A (RSV A) genomic RNA (gRNA) down to 1 aM under isothermal conditions, which is 100-fold more sensitive than regular NASBA.

A hairpin probe-mediated isothermal amplification method to detect target nucleic acid.

We herein describe Hairpin probe-mediated Isothermal Amplification (HIAmp), a novel isothermal method to detect a target nucleic acid. This method employs a hairpin probe (HP) designed to be opened through binding to the target nucleic acid. Upon opening of the HP, the primer binds to the free stem of the opened HP followed by its extension by DNA polymerase, consequently displacing and recycling the target nucleic acid to open another HP and producing an intermediate product (IP) containing a nicking site. The IP then continuously produces a trigger probe (TP), which subsequently initiates the isothermal amplification cycles in two separate ways by binding to either the intact HP or the overhang region of the IP. Through the following well-designed interconnected pathways, a large amount of final double-stranded DNA products (FPs) is produced and a high fluorescent signal is generated from the duplex-specific fluorescent dye, SYBR Green I. By employing this isothermal strategy, target DNA was very sensitively detected down to 64 zmol with the capability to discriminate the target DNA against non-specific DNAs. This work would provide remarkable insight into the design of a new DNA network enabling isothermal amplification.

Electrochemical Immunosensor for Human IgE Using Ferrocene Self-Assembled Monolayers Modified ITO Electrode.

The immunoglobulin E (IgE) level in serum is an important factor in the examination of allergy. Ferrocene (Fc)-modified self-assembled monolayers (SAMs) were placed on an indium tin oxide (ITO) electrode as a sensing layer for the detection of human IgE. The Fc moiety in the SAMs facilitated the electron transfer through the organic SAMs layer and electrocatalytic signal amplification. The electrochemical measurement was accomplished after the sandwich type immobilization of the receptor antibody, target human IgE, and enzyme conjugated secondary antibody. The enzyme product, p-aminophenol, was quantitatively analyzed by redox cycling via Fc. In addition, the electrochemical impedance spectroscopy (EIS) was investigated for the detection of IgE. The limit of detection (LOD), limit of quantification (LOQ), and dynamic range of the electrochemical sensor were 3 IU/mL, 10 IU/mL, and from 10 IU/mL to 100 IU/mL, respectively.

Label-free and washing-free alkaline phosphatase assay using a personal glucose meter.

We herein describe a personal glucose meter (PGM)-based method for a label-free and washing-free determination of alkaline phosphatase (ALP) activity, which relies on the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase to couple ALP activity with the amount of glucose. In principle, the presence of target ALP scavenges on adenosine 5'-triphosphate (ATP), a phosphate source for hexokinase-catalyzed reactions, and thus suppresses the ensuing cascade enzymatic reactions. As a result, the initial high amount of glucose is maintained and the amount of glucose, which is proportional to ALP activity, is simply measured by a hand-held PGM. Based on this novel strategy, we successfully determined the ALP activity down to 8.9 U/L with the high selectivity. In addition, the diagnostic capability of this method was demonstrated by reliably assaying the ALP activity in non-diluted human blood without any pretreatment steps.

A Personal Glucose Meter for Label-Free and Washing-Free Biomolecular Detection.

We developed a label-free and washing-free method for biomolecular detection using a personal glucose meter (PGM). ATP was selected as a model target, and cascade enzymatic reactions promoted by hexokinase and pyruvate kinase were adopted to link the amount of ATP to glucose that is detectable by a hand-held PGM. In principle, the presence of target ATP enables hexokinase to catalyze the conversion of glucose to glucose 6-phosphate by providing a phosphate group to glucose, and thus the amount of glucose is decreased in proportion to the amount of ATP. In addition, adenosine 5'-diphosphate (ADP), which is generated after hexokinase-catalyzed enzymatic reaction, is recovered to ATP by a pyruvate kinase enzyme. The regenerated ATP is again supplemented to catalyze multiple rounds of cascade enzymatic reactions, leading to signal amplification. As a result, the change of glucose amount that is inversely proportional to ATP amount is simply measured by a hand-held PGM. By employing this strategy, we successfully determined ATP down to 49 nM with high selectivity even in real samples such as tap water, human serum, and bovine urine. Importantly, the developed system does not require expensive modification and washing steps but is conveniently operated with a commercially available PGM, which would pave the way for the development of a simple and cost-effective sensing platform.

Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase.

We herein describe A novel strategy to accurately determine uracil DNA glycosylase (UDG) activity is described based on the finding that nicking endonuclease-assisted cleavage reaction can be regulated by the presence of abasic site. This strategy utilizes DNA probes rationally designed to contain uracil base at the cleavage site for nicking endonuclease, which is coupled to the isothermal nicking endonuclease amplification reaction (NEAR) method. In the absence of UDG, intact DNA probes generate a large number of double-stranded (ds) DNA products through the NEAR, but the presence of UDG that converts uracil base into abasic site suppresses nicking endonuclease activity and the subsequent NEAR. As a result, dsDNA products are not produced, which is simply monitored by the dsDNA specific fluorescence dye, SYBR green I. By employing this strategy, we sensitively determined the UDG activity down to 0.003 U mL-1 with high specificity over other base excision enzymes. In addition, the diagnostic capability of this method was successfully verified by reliably assaying UDG present in a human serum sample.