less Detection of Avian Influenza A Virus
Coupling of Fluorophore-DNA Barcode and Bead-based Immunoassay for PCRless Detection of Avian Influenza A Virus Cuong Cao,1 Raghuram Dhumpa,2 Dang Duong Bang,2 Zohreh Ghavifekr,2 Jonas Høgberg,2 and Anders Wolff 1 1
DTU-Nanotech, Department of Micro and Nanotechnology, Technical University of Denmark
2 DTU-VET, Laboratory of Applied Micro-nanotechnology, Department of Poultry, Fish, and Fur Animals, The National Veterinary Institute, Technical University of Denmark
May 12, 2009
Contents 1.
Introduction 1.1. Overview of Avian Influenza Virus 1.2. Basic concept of DNA Biobarcode-based Detection
2.
Experiment details 2.1. Overall Scheme of Fluorophore-DNA Barcode for Detection of Avian Influenza Virus 2.2. Results
3.
2
Summary and Conclusions
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1
1.1. Introduction: Overview of Avian Influenza Virus
HA (Hemagglutinin) NA (Neuraminidase) M2 ion channel RNA
Lipid layer
M1 matrix protein
Avian Influenza A Virus:
• Orthomyxoviridae family •Variable regions in the AIV genome are surface glycoproteins: hemagglutinin (H) and neuraminidase (N) → 16 H-subtypes (H1-H16) and 9 N-subtypes (N1-N9) 3
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.1. Introduction: Overview of Avian Influenza Virus (cont.) The spread of Avian Influenza Virus
Affected areas with confirmed cases of H5N1 avian influenza since 2003, status as of 06.05.2009. (Source : World Health Organization) 4
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2
1.1. Introduction: Overview of Avian Influenza Virus (cont.)
Cumulative Human Cases of and Deaths from H5N1 as of April 11, 2007
Cumulative Human Cases of and Deaths from H1N1 as of May 12, 2009
(Source : World Health Organization)
→ Threat for human health and enormous economic losses → Sensitive, specific and rapid detection of AIV is required 5
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.1. Introduction: Overview of Avian Influenza Virus (cont.) Main methods for diagnosis of viral infections
Madin-Darby canine kidney (MDCK) cell culture
Complement fixation (CF)
RT-PCR
6
DTU Nanotech, Technical University of Denmark
Hemagglutinin -inhibition (HI)
Immunoassays
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
3
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection Probe Design and Preparation
Science 2003, 301, 1884-1886 7
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Protein Detection and Barcode DNA Amplification and Identification
Science 2003, 301, 1884-1886 8
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
4
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Detection of PSA Protein Biomarker via DNA Biobarcode with 30 nm AuNP Probes
Science 2003, 301, 1884-1886 9
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Biobarcode-Based DNA Detection with PCR-like Sensitivity
J. AM. CHEM. SOC. 2004, 126, 5932-5933 10
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
5
2.1. Experiment details: Overall Scheme of Fluorophore-DNA Barcode for Detection of Avian Influenza Virus
AIV virus subtype H16N3 (HA 1:128, prepared by National Veterinary Institute, DTU-VET) Oligonucleotides Sequence (5’ → 3’) Oligo 1 Amino-C6-AAAAAAAAAA AGG AAG GTG TGG ACG ACG TCA AGT CAT CAT GGC C Oligo 2 Cy5-C GCC ATG ATG ACT TGA CGT CGT CCA CAC CTT CCT Forward primer AGA TGA GTC TTC TAA CCG AGG TCG Reverse primer TGC AAA AAC ATC TTC AAG TCT CTG
Oligonucleotide sequences used in the experiment
11
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2.2. Results Validation of the immunoprobes
→ The immunoprobes were prepared successfully and sufficient for catching the AIV
12
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
6
2.2. Results Detection of H16N3 AIV
Scanometric image showing fluorescence intensity of the microarrays corresponding to the diluted concentrations of H16N3 AIV
13
Logarithmic correlation of the spot intensity and diluted concentrations for the detection of H16N3 AIV (HA titre 1:128).
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2.2. Results Detection of H16N3 AIV by RT-PCR
DNA ladder
100
10-1
10-2
10-3
10-4
10-5
10-6
10-7
NC
PC
The virus strain was not recognized at the dilutions higher than 10-5 14
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
7
3. Summary and Conclusions An effective method using fluorophore-DNA barcode was described for the detection of AIV: 9The single immuno-recognition events were translated into multitude numbers of the Cy5-tagged DNA barcodes which could be quantitatively measured by a fluorescence readout device. 9H16N3 AIV strain could be detected with PCR-like sensitivity and wide dynamic range of concentration up to five orders of magnitude. 9Although simple in design and concept, the method introduced is PCRcomparably sensitive, less time-consuming (less than 2 hr for the entire detection), highly applicable, and can be carried out using simple laboratory equipments 9The assay is deserved itself as an alternative for surveillance and clinical detection of AIV outbreaks. 15
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
Acknowledgment This work was supported by the Danish Research Council for technology and production sciences (FTP) grant no. 274-05-0017.
Thank you for your attention !
16
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
8
DTU-Nanotech, Department of Micro and Nanotechnology, Technical University of Denmark
2 DTU-VET, Laboratory of Applied Micro-nanotechnology, Department of Poultry, Fish, and Fur Animals, The National Veterinary Institute, Technical University of Denmark
May 12, 2009
Contents 1.
Introduction 1.1. Overview of Avian Influenza Virus 1.2. Basic concept of DNA Biobarcode-based Detection
2.
Experiment details 2.1. Overall Scheme of Fluorophore-DNA Barcode for Detection of Avian Influenza Virus 2.2. Results
3.
2
Summary and Conclusions
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1
1.1. Introduction: Overview of Avian Influenza Virus
HA (Hemagglutinin) NA (Neuraminidase) M2 ion channel RNA
Lipid layer
M1 matrix protein
Avian Influenza A Virus:
• Orthomyxoviridae family •Variable regions in the AIV genome are surface glycoproteins: hemagglutinin (H) and neuraminidase (N) → 16 H-subtypes (H1-H16) and 9 N-subtypes (N1-N9) 3
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.1. Introduction: Overview of Avian Influenza Virus (cont.) The spread of Avian Influenza Virus
Affected areas with confirmed cases of H5N1 avian influenza since 2003, status as of 06.05.2009. (Source : World Health Organization) 4
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2
1.1. Introduction: Overview of Avian Influenza Virus (cont.)
Cumulative Human Cases of and Deaths from H5N1 as of April 11, 2007
Cumulative Human Cases of and Deaths from H1N1 as of May 12, 2009
(Source : World Health Organization)
→ Threat for human health and enormous economic losses → Sensitive, specific and rapid detection of AIV is required 5
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.1. Introduction: Overview of Avian Influenza Virus (cont.) Main methods for diagnosis of viral infections
Madin-Darby canine kidney (MDCK) cell culture
Complement fixation (CF)
RT-PCR
6
DTU Nanotech, Technical University of Denmark
Hemagglutinin -inhibition (HI)
Immunoassays
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
3
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection Probe Design and Preparation
Science 2003, 301, 1884-1886 7
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Protein Detection and Barcode DNA Amplification and Identification
Science 2003, 301, 1884-1886 8
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
4
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Detection of PSA Protein Biomarker via DNA Biobarcode with 30 nm AuNP Probes
Science 2003, 301, 1884-1886 9
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
1.2. Introduction: Basic concept of DNA Biobarcode-based Detection (cont.) Biobarcode-Based DNA Detection with PCR-like Sensitivity
J. AM. CHEM. SOC. 2004, 126, 5932-5933 10
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
5
2.1. Experiment details: Overall Scheme of Fluorophore-DNA Barcode for Detection of Avian Influenza Virus
AIV virus subtype H16N3 (HA 1:128, prepared by National Veterinary Institute, DTU-VET) Oligonucleotides Sequence (5’ → 3’) Oligo 1 Amino-C6-AAAAAAAAAA AGG AAG GTG TGG ACG ACG TCA AGT CAT CAT GGC C Oligo 2 Cy5-C GCC ATG ATG ACT TGA CGT CGT CCA CAC CTT CCT Forward primer AGA TGA GTC TTC TAA CCG AGG TCG Reverse primer TGC AAA AAC ATC TTC AAG TCT CTG
Oligonucleotide sequences used in the experiment
11
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2.2. Results Validation of the immunoprobes
→ The immunoprobes were prepared successfully and sufficient for catching the AIV
12
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
6
2.2. Results Detection of H16N3 AIV
Scanometric image showing fluorescence intensity of the microarrays corresponding to the diluted concentrations of H16N3 AIV
13
Logarithmic correlation of the spot intensity and diluted concentrations for the detection of H16N3 AIV (HA titre 1:128).
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
2.2. Results Detection of H16N3 AIV by RT-PCR
DNA ladder
100
10-1
10-2
10-3
10-4
10-5
10-6
10-7
NC
PC
The virus strain was not recognized at the dilutions higher than 10-5 14
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
7
3. Summary and Conclusions An effective method using fluorophore-DNA barcode was described for the detection of AIV: 9The single immuno-recognition events were translated into multitude numbers of the Cy5-tagged DNA barcodes which could be quantitatively measured by a fluorescence readout device. 9H16N3 AIV strain could be detected with PCR-like sensitivity and wide dynamic range of concentration up to five orders of magnitude. 9Although simple in design and concept, the method introduced is PCRcomparably sensitive, less time-consuming (less than 2 hr for the entire detection), highly applicable, and can be carried out using simple laboratory equipments 9The assay is deserved itself as an alternative for surveillance and clinical detection of AIV outbreaks. 15
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
Acknowledgment This work was supported by the Danish Research Council for technology and production sciences (FTP) grant no. 274-05-0017.
Thank you for your attention !
16
DTU Nanotech, Technical University of Denmark
Presentation name 17/04/2008 Cuong Cao, 12/05/2008
8
