Introducing Triplex Forming Oligonucleotide into Loop-Mediated Isothermal Amplification for Developing a Lateral Flow Biosensor for Streptococci Detection.

Loop-mediated isothermal amplification (LAMP) technology is extensively utilized for the detection of infectious diseases owing to its rapid processing and high sensitivity. Nevertheless, conventional LAMP signaling methods frequently suffer from a lack of sequence specificity. This study integrates a triplex-forming oligonucleotide (TFO) probe into the LAMP process to enhance sequence specificity. This TFO-LAMP technique was applied for the detection of Group B Streptococcus (GBS). The TFO probe is designed to recognize a specific DNA sequence, termed the TFO targeting sequence (TTS), within the amplified product, facilitating detection via fluorescent instrumentation or lateral flow biosensors. A screening method was developed to identify TFO sequences with high affinity to integrate TFO into LAMP, subsequently incorporating a selected TTS into an LAMP primer. In the TFO-LAMP assay, a FAM-labeled TFO is added to target the TTS. This TFO can be captured by an anti-FAM antibody on lateral flow test strips, thus creating a nucleic acid testing biosensor. The efficacy of the TFO-LAMP assay was confirmed through experiments with specimens spiked with varying concentrations of GBS, demonstrating 85% sensitivity at 300 copies and 100% sensitivity at 30,000 copies. In conclusion, this study has successfully developed a TFO-LAMP technology that offers applicability in lateral flow biosensors and potentially other biosensor platforms.

Development and optimization of a DNA aptamer to delay ß-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. ß-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against ß-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for ß-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'-O-methylation and a 3'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for ß-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of ß-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

Methods for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer Detection: Application of Differential Centrifugation and Annexin A5 Coated Beads.

Extracellular vesicles (EVs) contain abundant extracellular RNA (exRNA), which can be a valuable source of liquid biopsy. However, as various RNA species exist in different types of EVs, lack of detailed characterization of these RNA species and efficient collection methods limits the clinical application of exRNA. In the present study, we measured two mRNAs, CK19 and PCTK1; one lncRNA, MALAT1; and two miRNAs, miR21 and miR155, in different EV fractions separated by differential centrifugation or captured by magnetic beads coated with annexin A5 (ANX beads). The results showed that in a cultured medium, the majority of mRNA and lncRNA exist in larger EVs, whereas miRNA exist in both large and small EVs from the differential centrifugation fractions. All these RNA species exist in ANX beads captured EVs. We then used ANX beads to capture EVs in plasma samples from non-small-cell lung cancer patients and age-matched healthy volunteers. We found that the ANX bead capturing could efficiently improve RNA detection from human plasma, compared with direct extraction of RNA from plasma. Using ANX-bead capturing and reverse transcription and quantitative PCR, we detected significantly higher levels of CK19 mRNA, MALAT1 lncRNA, and miR155 miRNA in the plasma of lung cancer patients. These facts suggested the collection methods strongly affect the results of exRNA measurement from EVs, and that ANX beads can be a useful tool for detecting exRNA from plasma samples in clinical application.

Ultrafast DNA Amplification Using Microchannel Flow-Through PCR Device.

Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 µm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.

Targeted Sequencing of Circulating Cell Free DNA Can Be Used to Monitor Therapeutic Efficacy of Tyrosine Kinase Inhibitors in Non-small Cell Lung Cancer Patients.

BACKGROUND/AIM: Circulating tumor DNA (ctDNA) bears specific mutations derived from tumor cells. The amount of mutant ctDNA may reflect tumor burden. In this study, we detected epidermal growth factor receptor (EGFR) mutations in ctDNA as a monitoring marker for the response of non-small cell lung cancer (NSCLC) patients to tyrosine kinase inhibitors (TKIs). PATIENTS AND METHODS: Serial plasma samples from eight NSCLC patients during TKI treatment were collected. Libraries with barcoded adapters were constructed from ctDNA of these plasma samples using a PCR-based targeted DNA panel. The libraries were then sequenced for measuring EGFR mutations. In addition, carcinoembryonic antigen (CEA) was also measured in these patients. RESULTS: In six patients who suffered disease progression (PD), five had elevated EGFR mutation reads before PD. In the two patients who did not develop PD, EGFR mutations remained undetectable in their plasma. The CEA levels were higher than the cutoff value in most samples and had a poor correlation with disease status. CONCLUSION: The mutation count of tumor-specific mutations can be a monitoring marker of TKI treatment in NSCLC patients.

Transcriptomic Analysis in Liquid Biopsy Identifies Circulating PCTAIRE-1 mRNA as a Biomarker in NSCLC.

BACKGROUND/AIM: Circulating mRNA can be a useful source of cancer biomarkers. We took advantage of direct transcriptomic analysis in plasma RNA to identify novel mRNA markers for non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Plasma RNA from NSCLC patients and healthy individuals was profiled with cDNA-mediated annealing, selection, extension and ligation (DASL) microarrays. The microarray results were further validated in plasma RNA. RESULTS: Through RNA profiling and online database mining, four gene transcripts were filtered as candidate markers of NSCLC. After validation, the PCTAIRE-1 transcript was identified as a circulating mRNA marker. The diagnostic potential of PCTAIRE-1 was evaluated by receiver operating characteristic curve analysis, which gave a sensitivity and specificity of 60% and 85%, respectively. In addition, high plasma PCTK1 levels were also correlated with poor progression-free survival (p=0.008). CONCLUSION: Circulating mRNA can be profiled with the DASL assay. From the profile, PCTAIRE-1 RNA in the plasma we discovered as a novel diagnostic/prognostic biomarker and an indicator of poor survival in NSCLC patients.

Detection of Rare Somatic GNAS Mutation in McCune-Albright Syndrome Using a Novel Peptide Nucleic Acid Probe in a Single Tube.

McCune-Albright syndrome (MAS) is characterized by the triad of precocious puberty, café au lait pigmentation, and polyostotic fibrous dysplasia (FD) of bone, and is caused by post-zygotic somatic mutations-R201H or R201C-in the guanine nucleotide binding protein, alpha stimulating (GNAS) gene. In the present study, a novel peptide nucleic acid (PNA) probe with fluorescent labeling was designed to detect trace amounts of somatic mutant GNAS in a single tube reaction. The method was applied to screen GNAS mutations in six patients with MAS/FD. The results showed that the PNA probe assay could detect low abundant mutants in 200-fold excess of wild-type alleles. The GNAS mutation was found in three patients with severe disease (MAS) by using the assay. The other three patients with mild disease (having only FD) showed a wild-type result. This study has provided a simple method to detect trace amounts of GNAS mutants with high sensitivity in large amounts of wild-type DNA.

Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

A primer and probe set for detecting multiple types of EGFR exon 19 deletions.

EGFR exon 19 deletion is an important indicator for tyrosine kinase inhibitor treatment in non-small cell lung cancer. However, detection of exon 19 deletions faces a challenge: there are more than 30 types of mutations reported at the hotspot. Moreover, considering the application in body fluid samples, assays with high sensitivity and specificity are necessary for the detection of rare mutant alleles. Here, we describe a single tube reaction which could detect at least 29 types of exon 19 deletions with only an unlabeled peptide nucleic acid (PNA) clamp and a pair of DNA probes. The PNA clamp was used to inhibit amplification of wild-type templates; and the DNA probes were used to generate melting peaks for multiple types of mutations. Under optimal condition, the assay was able to detect as low as 0.01% mutant DNA in wild-type background, and had a limit of detection of 10 pg genomic DNA. Feasibility of the assay was tested in body fluid samples from lung cancer patients. The assay detected 100% and 60% of deletions in pleural effusions and plasma, respectively. We believe the present assay can be used in the clinical laboratories and has potential to be adapted for a microfluidic device.

A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing.

GeneGazer: A Toolkit Integrating Two Pipelines for Personalized Profiling and Biosignature Identification.

BACKGROUND: Next-generation sequencing provides useful information about gene mutations, gene expression, epigenetic modification, microRNA expression, and copy number variations. More and more computing tools have been developed to analyze this large quantity of information. However, to test and find suitable analytical tools and integrate their results is tedious and challenging for users with little bioinformatics training. In the present study, we assembled the computing tools into a convenient toolkit to simplify the analysis and integration of data between bioinformatics tools. MATERIALS AND METHODS: The toolkit, GeneGazer, comprises of two parts: the first, named Gaze_Profiler, was designed for personalized molecular profiling from next-generation sequencing data of paired samples; the other, named Gaze_BioSigner, was designed for the discovery of disease-associated biosignatures from expressional and mutational profiles of a cohort study. RESULTS: To demonstrate the capabilities of Gaze_Profiler, we analyzed a pair (colon cancer and adjacent normal tissues) of RNA-sequencing data from one patient downloaded from the Sequencing Read Archive database and used them to profile somatic mutations and digital gene expression. In this case, alterations in the RAS/RAF/MEK/ERK signaling pathway (activated by KRAS G13D mutation) and canonical WNT signaling pathway (activated by truncated APC) were identified; no EGFR mutation or overexpression was found. These data suggested a limited efficacy of cetuximab in the patient. To demonstrate the ability of Gazer_BioSigner, we analyzed gene-expression data from 192 cancer tissues downloaded from The Cancer Genome Atlas and found that the activation of cAMP/PKA signaling, OCT-3/4 and SRF were associated with colon cancer progression and could be potential therapeutic targets. CONCLUSION: GeneGazer is a reliable and robust toolkit for the analysis of data from high-throughput platforms and has potential for clinical application and biomedical research.

Spin-coated Au-nanohole arrays engineered by nanosphere lithography for a Staphylococcus aureus 16S rRNA electrochemical sensor.

The nanopatterning of gold nanoparticle (AuNP) arrays on an indium tin oxide (ITO) electrode using efficient and low-cost methods is described. This process used nanosphere lithography (NSL) encompassing the deposition of monolayered Polystyrene (PS) followed by a convective self-assembly drop coating protocol onto the ITO substrate that further acted as the mask after the AuNP assembly. The results showed that spin-coating allowed AuNPs to follow the contour and adhere to the PS nanospheres. The final products, after etching the PS, generated a highly ordered Au-nanohole array on an ITO substrate. The Au-nanohole arrays on the ITO electrode provided a greater surface area and successfully enhanced the peak current of electrochemical measurements by 82% compared with bare ITO and was used to detect Staphylococcus aureus 16S rRNA hybridization. In contrast to non-templated AuNP structures, the Au-nanohole arrays on the ITO electrode contributed to an optimum sensitivity improvement in DNA hybridization detection by 23%, along with an impressive limit of detection (LOD) of 10 pM. The high specificity of this distinguished structure was also achieved in the hybridization measurements of multi-analyte pathogens. These findings indicate that the combination of PS nanosphere lithography, followed by the spin-coating of AuNPs, leads to an inexpensive and simple engineering process that effectively generates uniform Au-nanohole arrays on ITO, which provides a greater surface area to optimize the electrochemical performance of the DNA biosensor.

Development of a magnetic bead-based method for the collection of circulating extracellular vesicles.

Cells release different types of extracellular vesicles (EVs). These EVs contain biomolecules, including proteins and nucleic acids, from their parent cells, which can be useful for diagnostic applications. The aim of this study was to develop a convenient procedure to collect circulating EVs with detectable mRNA or other biomolecules. Magnetic beads coated with annexin A5 (ANX-beads), which bound to phosphatidylserine moieties on the surfaces of most EVs, were tested for their ability to capture induced apoptotic bodies in vitro and other phosphatidylserine-presenting vesicles in body fluids. Our results show that up to 60% of induced apoptotic bodies could be captured by the ANX-beads. The vesicles captured from cultured media or plasma contained amplifiable RNA. Suitable blood samples for EV collection included EDTA-plasma and serum but not heparin-plasma. In addition, EVs in plasma were labile to freeze-and-thaw cycles. In rodents xenografted with human cancer cells, tumor-derived mRNA could be detected in EVs captured from serum samples. Active proteins could be detected in EVs captured from ascites but not from plasma. In conclusion, we have developed a magnetic bead-based procedure for the collection of EVs from body fluids and proved that captured EVs contain biomolecules from their parent cells, and therefore have great potential for disease diagnosis.

Programming a nonvolatile memory-like sensor for KRAS gene sensing and signal enhancement.

A programmable field effect-based electrolyte-insulator-semiconductor (EIS) sensor constructed with a nonvolatile memory-like structure is proposed for KRAS gene DNA hybridization detection. This programmable EIS structure was fabricated with silicon oxide (SiO2)/silicon nitride (Si3N4)/silicon oxide on a p-type silicon wafer, namely electrolyte-oxide-nitride-oxide-Si (EONOS). In this research, voltage stress programming from 4 to 20V was applied to trigger holes confinement in the nitride-trapping layer that, consequently, enhances the DNA attachment onto the sensing surface due to additional electrostatic interaction. Not solely resulting from the higher DNA load, the programming may affect the orientation of the DNA that finally contributes to the change in capacitance. Findings have shown that a higher voltage program is able to increase the total capacitance and results in ~3.5- and ~5.5-times higher sensitivities for a series of concentrations for complementary DNA and wild type versus mutant DNA hybridization detection, respectively. Overall, it has been proven that the voltage program on the nonvolatile memory-like structure of EONOS is a notable candidate for genosensor development, scoping the diagnosis of a single nucleotide polymorphism (SNP)-related disease.

Circulating Messenger RNA Profiling with Microarray and Next-generation Sequencing: Cross-platform Comparison.

BACKGROUND: Circulating mRNA is a less invasive and more easily accessed source of samples for biomedical research and clinical applications. However, it is of poor quality. We explored and compared the ability of two high-throughput platforms for the profiling of circulating mRNA regarding their ability to retrieve useful information out of this type of samples. MATERIALS AND METHODS: Circulating mRNAs from three non-small cell lung cancer patients and three healthy controls were analyzed by the cDNA-mediated annealing, selection, extension, and ligation (DASL) assay and high-throughput RNA sequencing (RSEQ). Twelve genes were selected for further confirmation by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: The overall expression profiles derived from the two platforms showed modest-to-moderate correlation. Genes with higher expression levels had higher cross-platform concordance than those of medium- and low-expression levels. In addition, the pathway signatures identified by gene set enrichment analysis from both platforms were in agreement. The RT-q PCR results for the selected genes correlated well with that of RSEQ. CONCLUSION: Genes with higher expression levels have cross-platform concordance and can be potential biomarkers. Furthermore, RSEQ is a better tool for profiling circulating mRNAs.

Detection of low copies of drug-resistant influenza viral gene by a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.

Influenza virus infection causes endemics almost yearly and pandemics occasionally. Although antivirals are available for the clinical treatment of influenza virus infection, the emergence of a drug-resistant virus has reduced the effectiveness of therapy and prophylaxis. Therefore, the timely detection of drug-resistant influenza viruses is important. A single-tube reaction using peptide nucleic acid (PNA) as both a polymerase chain reaction (PCR) clamp and a sensor probe was established to detect the low numbers of copies of viral genes that carry the resistant marker. Influenza A H1N1 viruses resistant to a clinically used antiviral, amantadine, are selected for the experimental design. The PNA-mediated reverse transcription-PCR detected 10 copies/µL of RNA from the resistant strain among 2 × 10(4) copies/µL of RNA from the sensitive strain. A rapid and sensitive method was established for detecting low numbers of drug-resistant genes of the influenza virus. The assay would help to monitorthe emergence of adrug-resistant influenza virus.

Integrating solid-state sensor and microfluidic devices for glucose, urea and creatinine detection based on enzyme-carrying alginate microbeads.

A solid-state sensor embedded microfluidic chip is demonstrated for the detection of glucose, urea and creatinine in human serum. In the presented device, magnetic powder-containing enzyme-carrying alginate microbeads are immobilized on the surface of an electrolyte-insulator-semiconductor (EIS) sensor by means of a step-like obstacle in the microchannel and an external magnetic force. The sample is injected into the microchannel and reacts with the enzyme contained within the alginate beads; prompting the release of hydrogen ions. The sample concentration is then evaluated by measuring the resulting change in the voltage signal of the EIS sensor. The reaction time and alginate bead size are optimized experimentally using a standard glucose solution. The experimental results show that the device has a detection range of 2-8mM, 1-16mM and 10(-2)-10mM for glucose, urea and creatinine, respectively. Furthermore, it is shown that the device is capable of sequentially measuring all three indicators in a human serum sample. Finally, it is shown that the measured values of the glucose, urea and creatinine concentrations obtained using the device deviate from those obtained using a commercial kit by just 5.17%, 6.22% and 13.53%, respectively. This method can be extended to sequentially measure multiple blood indicators in the sample chip by replacing different types of enzyme in alginate bead and can address the enzyme preservation issue in the microfluidic device. Overall, the results presented in this study indicate that the microfluidic chip has significant potential for blood monitoring in point-of-care applications.

Sensitive detection of unlabeled oligonucleotides using a paired surface plasma waves biosensor.

Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides' relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization. PSPWB measures the demodulated amplitude of the heterodyne signal in real time. In the meantime, the ratio of the amplitudes between the detected output signal and reference can reduce the excess noise from the laser intensity fluctuation. Also, the common-path propagation of p and s waves cancels the common phase noise induced by temperature variation. Thus, a high signal-to-noise ratio (SNR) of the heterodyne signal is detected. The sequence specificity of oligonucleotide hybridization ensures that the platform is precisely discriminating between target and non-target oligonucleotides. Under optimized experimental conditions, the detected heterodyne signal increases linearly with the logarithm of the concentration of target oligonucleotide over the range 0.5-500 pM. The detection limit is 0.5 pM in this experiment. In addition, the non-target oligonucleotide at concentrations of 10 pM and 10nM generated signals only slightly higher than background, indicating the high selectivity and specificity of this method. Different length of perfectly matched oligonucleotide targets at 10-mer, 15-mer and 20-mer were identified at the concentration of 150 pM.

Monitoring triplex DNA formation with fluorescence resonance energy transfer between a fluorophore-labeled probe and intercalating dyes.

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.

Detection of pandemic (H1N1) 2009 influenza virus by allele discrimination.

BACKGROUND: The pandemic (H1N1) 2009 has become a threat of public health. To manage rapidly increased infections and disease control, a simple and reliable first-line screening test for viral infection is on urgent demand. METHODS: Through comprehensive bioinformatics analysis, a single nucleotide polymorphism in nucleoprotein gene which differentiates swine lineage virus and human seasonal H1N1 virus was selected as target. A TaqMan probe-based allele discrimination analysis was designed to analyze clinical samples. In total, 93 clinical specimens and 39 viral isolates were used to test the assay efficacy. Traditional viral culture and molecular analysis were used as gold standard. RESULTS: The testing results showed that the established assay has high sensitivity and specificity (92% and 100%) for pandemic (H1N1) 2009. The assay could detect as low as 5 copies of NP gene of pandemic (H1N1) 2009 or 2 viral particles of human seasonal H1N1. CONCLUSION: This assay can be used as a first-line screening and confirmation test for pandemic (H1N1) 2009 virus and human seasonal flu in one-tube reaction. The assay can serve as a convenient method to reduce the burden of PCR manipulation for diagnostic laboratories when large amount of samples need to be analyzed in a short time.