Assays for urinary biomarkers of oxidatively damaged nucleic acids

Abstract

The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme-linked immunosorbent assay). The major analytical challenge is specificity. The best combination of selectivity and speed of analysis can be obtained by liquid chromatography coupled with tandem mass spectrometric detection. This, however, is also the most demanding technique with regard to price, complexity and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from the nucleotide pools is most probably minimal, if existing. Recent investigation on RNA oxidation has shown conditions where RNA oxidation but not DNA oxidation is prominent, and while investigation on DNA is of huge interest, RNA oxidation may be overlooked. The methods for analyzing oxidized deoxynucleosides can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA.     

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Quantification of 8-oxo-guanine and guanine as the nucleobase, nucleoside and deoxynucleoside forms in human urine by high-performance liquid chromatography–electrospray tandem mass spectrometry

Oxidative DNA damage, linked pathogenically to a variety of diseases such as cancer and ageing, can be investigated by measuring specific DNA repair products in urine. Within the last decade, since it was established that such products were excreted into urine, progress in their analysis in urine has been limited. Guanine is the DNA base most prone to oxidation. We present a method for determination of the urinary 8-hydroxylated species of guanine, based on direct injection of urine onto a high-performance liquid chromatography (HPLC)–tandem mass spectrometry system. The analysis covers the 8-hydroxylated base, ribonucleoside and deoxynucleoside, and the corresponding non-oxidised species. Without pre-treatment of urine the detection limits for the nucleobases are ~2 nM (50 fmol injected) and for the nucleosides ~0.5 nM (12.5 fmol injected). Previously, liquid chromatography of the nucleobases has been problematic but is made possible by low-temperature reverse-phase C18 chromatography, a method that increases retention on the column. In the case of the nucleosides, retention was almost total and provides a means for on-column concentration of larger urine samples and controlled high peak gradient elution. The total excretion of 8-hydroylated guanine species was 212 nmol/24 h. The oxidised base accounted for 64%, the ribonucleoside for 23% and the deoxynucleoside for 13%, indicating substantial oxidation of RNA in humans. In rat urine, excretion of the oxidised base was more dominant, the percentages of the oxidised base, ribonucleoside and deoxynucleosides being 89, 8 and 3%. This finding is at odds with previous reports using immunoaffinity pre-purification and HPLC–electrochemical detection analysis. The developed method now makes it possible to measure oxidative nucleic acid stress to both RNA and DNA in epidemiological and intervention settings, and our findings indicate a substantial RNA oxidation in addition to DNA oxidation. The small volume needed also makes the method applicable to small experimental animals.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.     

Facts and artifacts in the measurement of oxidative base damage to DNA

This short survey is aimed at critically evaluating the main available methods for measuring oxidative base damage within cellular DNA. Emphasis is placed on separative methods which are currently widely applied. These mostly concern high performance liquid chromatography (HPLC) and gas chromatography (GC) associated with sensitive detection techniques such as electrochemistry (EC) and mass spectrometry (MS). In addition, the comparison is extended to 32p-postlabeling methods, immunoassays and measurement of two main classes of oxidative DNA damage within isolated cells. It may be concluded that the HPLC-electrochemical detection (ECD) method, even if restricted to the measurement of only a few electroactive oxidized bases and nucleosides, is the simplest and safest available method at the moment. In contrast, the more versatile GC-MS method, which requires a HPLC pre-purification step in order to prevent artifactual oxidation of overwhelming normal bases to occur during derivatization, is more tedious and its sensitivity may be questionable. Alternative simpler procedures of background prevention for the GC-MS assay, which, however, remain to be validated, include low-temperature for derivatization and addition of antioxidants to the silylating reagents. Interestingly, similar levels of 8-oxo-7,8-dihydroguanine were found in cellular DNA using HPLC-ECD, HPLC-MS/MS and HPLC/32P-postlabeling methods. However, it should be noted that the level of cellular 8-oxodGuo, thus determined, is on average basis 10-fold higher than that was inferred for more indirect measurement involving the use of DNA repair enzymes with methods on isolated cells. Further efforts should be made to resolve this apparent discrepancy. In addition, the question of the biological validation of the non-invasive measurement of oxidized bases and nucleosides in urine is addressed.     

Detection and interpretation of 8-oxodG and 8-oxoGua in urine,plasma and cerebrospinal fluid

Background

DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mainly on degradation.

Scope of review

DNA and RNA oxidations are measured by very similar techniques. The scope of this review is to highlight the preferred methods of measurement of oxidized nucleic acid metabolites, to highlight novel findings particularly in RNA oxidation, and to present the interpretation of the measurements.

Major conclusions

Tissue levels are snap-shots of the level in a specific organ or cell system and reflect the balance between formation rate and elimination rate (repair), and must be interpreted as such. Urinary excretion is a global measure of oxidative stress in an organism and is therefore best suited for situations or diseases where large parts or the entire organism is stressed by oxidation. It represents the body average rate by which either RNA or DNA is oxidized and is interpreted as oxidative stress. Oxidations of RNA and DNA precursors have been demonstrated and the quantitative importance is debated.

General significance

Careful experimental designs and appropriate choice of methodology are paramount for correct testing of hypotheses related to oxidative stress, and pitfalls are plentiful. There is accumulating evidence that DNA oxidation is associated with disease, particularly cancer, and recent evidence points at an association between RNA oxidation and neurodegenerative diseases and diabetes. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

Molecular beacons: nucleic acid hybridization and emerging applications.

Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.     

Electric chips for rapid detection and quantification of nucleic acids

A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.     

Quantitative,multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system

Escherichia coli, Salmonella, Listeria monocytogenes and Campylobacter jejuni are bacterial pathogens commonly implicated in foodborne illnesses. Generally used detection methods (i.e., culture, biochemical testing, ELISA and nucleic acid amplification) can be laborious, time-consuming and require multiple tests to detect all of the pathogens. Our objective was to develop rapid assays to simultaneously detect these four organisms through the presence of antigen or DNA using the Luminex LabMAP system. For nucleic acid detection, organism-specific capture probes corresponding to the 23S ribosomal RNA gene (rrl) were coupled covalently to LabMAP microspheres. Target molecules included synthetic complementary oligonucleotides and genomic DNA isolated from ATCC type strains or other well-characterized strains of each organism. Universal PCR primers were designed to amplify variable regions of bacterial 23S ribosomal DNA, yielding biotinylated amplicons of 86 to 109 bp in length. Varying quantities of targets were hybridized to the combined microsphere sets, labeled with streptavidin-R-phycoerythrin and analyzed on the Luminex(100) system. Results of nucleic acid detection assays, obtained in 30 to 40 min following amplification, correctly and specifically identified each bacterial species with a detection sensitivity of 10(3) to 10(5) genome copies. Capture-sandwich immunoassays were developed with organism-specific antibodies coupled to different microsphere sets. Microspheres were incubated with organism-specific standards and reactivity was assessed with biotinylated detection antibodies and streptavidin-R-phycoerythrin. In the immunoassays, microsphere-associated fluorescence was organism concentration dependent with detectable response at < or = 1000 organisms/ml and with no apparent cross-reactivity. We have demonstrated that the Luminex LabMAP system is a rapid, flexible platform capable of simultaneous, sensitive and specific detection of pathogens. The practical significance of this multiplexing approach would be to provide more timely, economical and comprehensive information than is available with conventional isolation and identification methodologies.     

Locked nucleic acid flow cytometry-fluorescence in situ hybridization (LNA flow-FISH): a method for bacterial small RNA detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination, cellular identification and gene expression, monitoring viral multiplication in infected cells, and bacterial community analysis and enumeration. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (T(m)) of these analogs for natural nucleic acids. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes and have been successfully used to detect eukaryotic mRNA and viral RNA in mammalian cells. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).     

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.     

The effects of phenolic microbial metabolites on the activities of mitochondrial enzymes

The effects of microbial phenolic metabolites on the activities of enzymes of the tricarboxylic acid cycle were investigated in isolated mitochondria. The detection of metabolites of the tricarboxylic acid cycle in the blood of patients with sepsis as potential biomarkers of mitochondrial dysfunction was investigated. We found that microbial phenolic metabolites possess an inhibitory effect on the activity of dehydrogenases, as determined by the reduction of dichlorophenolindophenol and nitroblue tetrazolium in liver mitochondria and liver homogenates. The effect was more pronounced in the case of the oxidation of NAD-dependent substrates than succinate oxidation, as well as at lower concentrations of microbial metabolites than in the case of inhibition of respiration. Using gas chromatography coupled with mass spectrometry it was shown that the content of the tricarboxylic acid cycle metabolites is lower in the blood of patients with sepsis as compared to healthy donors. Our data demonstrate that microbial phenolic acids can significantly contribute to mitochondrial dysfunction and to metabolic suppression, both of which are characteristic of these pathologies.     

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.     

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

Exemestane is an irreversible aromatase inhibitor used for anticancer therapy. Unfortunately, this drug is also misused in sports to avoid some adverse effects caused by steroids administration. For this reason exemestane has been included in World Anti-Doping Agency prohibited list. Usually, doping control laboratories monitor prohibited substances through their metabolites, because parent compounds are readily metabolized. Thus metabolism studies of these substances are very important. Metabolism of exemestane in humans is not clearly reported and this drug is detected indirectly through analysis of its only known metabolite: 17β-hydroxyexemestane using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled to mass spectrometry (GC-MS). This drug is extensively metabolized to several unknown oxidized metabolites. For this purpose LC-MS/MS has been used to propose new urinary exemestane metabolites, mainly oxidized in C6-exomethylene and simultaneously reduced in 17-keto group. Urine samples from four volunteers obtained after administration of a 25mg dose of exemestane were analyzed separately by LC-MS/MS. Urine samples of each volunteer were hydrolyzed followed by liquid-liquid extraction and injected into a LC-MS/MS system. Three unreported metabolites were detected in all urine samples by LC-MS/MS. The postulated structures of the detected metabolites were based on molecular formulae composition obtained through high accuracy mass determination by liquid chromatography coupled to hybrid quadrupole-time of flight mass spectrometry (LC-QTOF MS) (all mass errors below 2ppm), electrospray (ESI) product ion spectra and chromatographic behavior.     

Extracting nucleic acids from activated sludge which reflect community population diversity

Critical to most studies in molecular microbial ecology is the application of DNA/RNA extraction methods which can reveal the true level of population biodiversity present in samples from the community under investigation. Activated sludge communities have been studied extensively using molecular methods, but rarely have the nucleic acid isolation methods applied been assessed for their ability to achieve this. This study compares eight published RNA and DNA extraction protocols and one commercially available DNA isolation kit for their capacity to provide high quality nucleic acids that reflect the community composition. Each method was assessed on the basis of nucleic acid yield, purity and integrity, and the ability to provide PCR amplifiable RNA and DNA from known marker populations that varied in their resistance to nucleic acid extraction. Only three consistently provided DNA from each of the marker populations known to be present in the samples from fluorescence in situ hybridisation analysis. The failure of the other methods emphasises the need to validate all DNA/RNA extraction protocols. It is recommended that several validated extraction methods be used and the extracts pooled to further minimise any risk of bias.     

Review on the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its phase I and phase II metabolites in biological matrices, foodstuff and beverages

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), has been shown to be carcinogenic in rodents, mice and rats. Following phase I N-hydroxylation and phase II esterification PhIP exerts its carcinogenic effect by binding to DNA purines. Quantitative and qualitative analysis of its bioactivated metabolites as well as it detoxification products is important in studying its biological effects and inter- and intra-individual exposures. A review is presented with an extensive coverage of publications specifically reporting on the analysis of PhIP and its phase I and II metabolites in biological matrices, foodstuff and beverages. Analytical techniques such as liquid and gas chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection) were mostly applied. We conclude that since the initial identification of PhIP in 1986 a large set of assays has been developed for the analysis of PhIP and its phase I and phase II metabolites in a wide range of matrices, these included food products and biological samples such as plasma, urine and faeces. In addition, it was shown that numerous metabolites were recovered and identified. Thus, we conclude that liquid chromatography coupled to mass spectrometry is clearly the method of choice for sensitive qualitative as well as quantitative analysis with high selectivity and reaching lower quantification levels in the sub pg/mL range. The main aim of this review is that it can be used by other researchers as a resource for method development and optimization of analytical methods of PhIP and its carcinogenic or detoxification products.     

Development Of A Nucleic Acid Lateral Flow Strip For Detection Of Hepatitis C Virus (Hcv) Core Antigen

The object of this study was to develop a simple, rapid, specific, and highly sensitive method to detect HCV core antigen. A nucleic acid aptamer was designed with the high specificity and sensitivity in a nucleic acid lateral flow strip to compete with HCV core antigen and DNA probes. The lower detection limit of the test strip was calculated to be 10 pg/mL with the scanner and 100 pg/mL with naked eyes. Results showed that there were no cross-interactions with other proteins such as HCV NS3, E1/E2 antigens, HIV p24 antigens, or BSA proteins (HCV unrelated protein). When the viral load exceeded 10⁴ copies/mL, the positive coincidence rates of ELISA and strip detection, when compared with the HCV RNA assay, were 98.44% and 97.28%, respectively. The results indicated that the ELISA detection and strip assay were in good agreement with the measured value. The results indicated that a nucleic acid lateral flow strip was a simple, rapid, specific, highly sensitive, and cost-effective field-based method for detecting HCV core antigen. The strip assay is an acceptable alternative to diagnose HCV core antigen and to investigate its epidemiology in clinical laboratories lacking specialized equipment and skills.