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Development of Electronic Microarray for Simultaneous Detection

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Development of Electronic Microarray for Simultaneous Detection Powered By Docstoc
					  Development of Electronic Microarray for
    Simultaneous Detection and Typing of
Avian Influenza and Newcastle Disease Viruses



                    Samuel Ohene-Adjei

       Canadian Food Inspection Agency. Lethbridge AB

                        CAHLN 2010
Avian Influenza
Outbreaks of HPAIV
1983     Pennsylvania      H5N2      17 million birds mostly chicken and turkeys
2003     Netherlands       H7N7      Virus isolated in 241 farms
                           30 million birds were culled
2004     Canada            H7N3      40 Commercial farms
                           19 million culled (Fraser Valley, BC)



Highly infectious
High mortality and morbidity rates
Transmission - close contact
Incubation period – within a few days
Economically devastating
16 HA and 9 NA subtypes
Newcastle Disease virus
2000-            Mexico - Chicken
2002-2003        USA (S. California, Nevada, Arizona, Texas)- Game fowl
2002             California (USA) – Transmission to commercial farms
                 3.5 million birds depopulated
9 Serotypes but 1 serotype associated with defined disease
Remains infectious
        in bone marrow and muscle 6 months at -20ºC
        in infected egg for over 1 year at 4ºC
Highly contagious – incubation period 4-6 days
Economically devastating (HP)
Diagnosis
Confirmatory lab diagnosis needed



Complications in veterinary diagnosis:

AIV and NDV may present same clinical symptoms

Possibility of mixed infection of AIV and NDV




Objectives
•To simultaneously detect AIV and NDV
•To discriminate between high and low pathogenic NDV
              Overview of assay development

   SAMPLE


                               PCR #2                  PCR #3
                               AIV-HA                  AIV-NA
 PCR #1
- AIV-Matrix (M)
- NDV-Matrix (M)
- NDV-Fusion (F)                 NC #2              NC #3
NC #1                       AIV-HA subtyping   AIV-NA subtyping
- AIV and NDV detection
- NDV pathotyping            Specificity Experiments
                             Multiplex RT-PCR
                             Microarray
                             42 AIV lab strains
                             22 NDV strains
               AIV 19    NEG
AIV 37         AIV 20    AIV 2
AIV 38         AIV 21    AIV 3
               AIV 22    AIV 4
AIV 39
               AIV 23    AIV 5
AIV 40
               AIV 24    AIV 6
AIV 41                   AIV 7
               AIV 25
AIV 42                   AIV 8
               AIV 26
AIV 43                   AIV 9
               AIV 27
NDV 1          AIV 28    AIV 10
                                  AIV




NDV 4          AIV 29    AIV 11
NDV 6          AIV 30    AIV 12
               AIV 31    AIV 13
               AIV 32    AIV 14
               AIV 33    AIV 15
               AIV 34    AIV 16
               AIV 35    AIV 17
               AIV 36    AIV 18




                        NEG
         E4
                        NDV-1
         E5
                        NDV-2
         E6
                        NDV-3
         E7
                        NDV-4
         E8
                        NDV-6
         E9
                        D10
         E10
                                  NDV




                        D11
         E11
                        D12
         E12
                        E1
         F1
                        E2
         F2
                        E3
                                        Gel images of RT-PCR amplified cDNA from lab strains
                         Electronic Microarray

•   Electrophoretically driven printing of probes and
    minimal amplicon hybridization time

•   400 independently activated test sites
                      Printing
                Hybridization
                 Reporting


 Biotinylated                      Amplicon
                                    Wash
Capture Probe




                +          +
           Positive     Positive
           Charge       Charge
                                                         probes
                                                         Detection




                                                        AIV M-007 deg

                                                        AIV-M-008 deg

                                                        NDV-M-01

                                                        NDV-M-10
                                                        FMD-AT-23




         22 NDV strains                41 AIV strains
                                                        probes
                                                        Pathotyping




                                                        NDV1-HP
                                                        NDV-HP-1
                                                        NDV-HP-2
                                                        NDV-HP-3
                                                        NDV-HP-4
                                                        NDV-HP-5
                                                        NDV-HP-6
                                                        NDV-HP-7
                                                        NDV-HP-8
                                                        NDV-HP8-VC22
                                                        NDV-LP8-NC22
                                                        NDV-LP-4
                                                        NDV-LP-8
                                                        FMD- AT-23




14 LP NDV strains
                    8 HP NDV strains   41 AIV strains
                                                                        Heat map – P:N data. Probe specificity to AI and NDV
                                                                   Sensitivity
             Detection probe
                                                                                              Pathotyping probe

            Sensitivity of detection probe to DYH Am azon (HP)                                       Sensitivity of HP probe

      25                                                                       25

      20                                                                       20     DYH Amazon (HP)                           B2 (LP)

      15                                                                       15




                                                                         P:N
P:N




      10                                                                       10

                                                                               5
      5

                                                                               0
      0
                                                                                       HP 3            FMD-AT-23               HP3           FMD-AT-23
                      NDV-M-10                      FMD-AT-23
                                                                                                                   Probe
                                      Probe

               10^5    10^4   10^3   10^2   10^1   10^0   10^-1   NTC                10^5     10^4   10^3   10^2    10^1   10^0      10^-1   10^-2   NTC




           Limit of detection 103 TCID50 /ml                                        Limit of detection 103 TCID50 / ml


           Limit of detection for NDV B2 using detection probe 101 TCID50/ml
                                                                        pathotyping probe 102 TCID50 / ml
                                  NDV Clinical Testing
  Clinical samples                                                   Bird 642 Cloacal swab



  10 birds infected with APMV-1 Amazon
                                                     20
                                                     18
                                                     16

  Sampling: Swabs: oropharynx, cloaca                14
                                                     12
                                                                                                         1dpi




                                               P:N
                                                                                                         2 dpi
                                                     10
                                                                                                         3 dpi
              Dpi: 1, 2, 3                            8
                                                      6
                                                                                                         NTC

                                                      4
  Total = 60 samples                                  2
                                                      0
                                                          NDV-M-01        NDV-HP-3           FMD-AT-23
  Field negative samples = 40                                               Probe




Comparison of Microarray to RT-PCR            Samples not detected

                         Cloaca               • RT-PCR Ct values close to cutoff (36)

        DPI      Array       RT-PCR           • Faint bands in PCR for array

          1         7             9           • Results highly comparable with

          2        10             10                 RT-PCR
          3         7             10
Avian HA and NA subtyping                        AIV Heat map of P:N ratio data




                                                       N2-014long1
                                                   Probes




                                                       NA6-2(qiu)



                                                       Neg.Cont.
40/41 strains correctly HA subtyped




                                                       N1-021
                                                       N1-026
                                                       N2-008

                                                       N3-002
                                                       N4-022
                                                       N5-017

                                                       N7-005
                                                       N8-015
                                                       N9-022
                                                                       NA Specificity

All 41 strains correctly NA subtyped
                                                                     N1
Sensitivity: comparable to real time PCR data

Limit of detection of NA assay 43 - 2320 EID50

Limit of detection of HA assay 4 - 23 EID50
                                                                     N2




                                                                               Strains
 Summary
                                                                     N3
 •   Simultaneous detection of AIV and NDV
                                                                     N4
     and further pathotype NDV
                                                                     N5
 •   No cross reactions observed                                     N6

 •   Assay useful for single and mixed                               N7

     infection
 •   Results highly comparable to RT-PCR                             N8


                                                                     N9

                                                                     NTC
CFIA, Lethbridge          CFIA, NCFAD, Winnipeg
Oliver Lung               John Pasick
Dirk Deregt *             Yohannes Berhane
Tara Furukawa-Stoffer     Animal and Technical Staff
Kimberley Burton Hughes   Alfonso Clavijo*
Mathew Fisher
Anne Beeston*
Kristen Hahn*             IAH, Pirbright, UK
Jennifer Geddes*          Donald King
Billy Mauro*              Scott Reid

Nexogen, Inc.             Funding:
Dalibor Hodko
                          CRTI (The Chemical, Biological, Radiological
* Former members          or Nuclear Research and Technology Initiative)




                              CFIA Lethbridge Laboratory
Outline
Outbreaks

Electronic Microarray

Overview of Avian HC Assay: Design and Development

Results
       NDV/AIV Multiplex RT-PCR Design and
                 Development


     280 bp

                   AIV Matrix (M) gene
1                                                      1027


          400 bp

                      NDV Matrix (M) gene
1                                                        1097

       344 bp

                                 NDV Fusion (F) gene
1                                                               1662
    Cleavage Region
  Electronic Microarray

• Electrophoretically driven printing and
  hybridization
• 400 independently activated test sites
                                                           (Movie here)


                                                        Time (seconds)    Time (hours)
                                                        Electronic          Passive


                             50 m diameter test site


 Array

 Hydrogel Permeation Layer
  Containing Streptavidin


         CMOS Chip              Platinum
                              Microelectrode
     Heat map – P:N data. Reactivity of probes to AI and NDV

Detection

            AIV M-007 deg

                            AIV-M-008 deg




                                                                                                                                                                                                                                                         NDV-HP8-VC22
                                                                                                                                         Pathotyping




                                                                                                                                                                                                                                                                        NDV-LP8-NC22
                                                                  FMD-AT-23
                                            NDV-M-01

                                                       NDV-M-10




                                                                                                                                                                                                                                                                                                             FMD- AT-23
probes




                                                                                                                                                                 NDV-HP-1
                                                                                                                                                                            NDV-HP-2
                                                                                                                                                                                       NDV-HP-3
                                                                                                                                                                                                  NDV-HP-4
                                                                                                                                                                                                             NDV-HP-5
                                                                                                                                                                                                                        NDV-HP-6
                                                                                                                                                                                                                                   NDV-HP-7
                                                                                                                                                                                                                                              NDV-HP-8




                                                                                                                                                                                                                                                                                       NDV-LP-4
                                                                                                                                                                                                                                                                                                  NDV-LP-8
                                                                                                                                                       NDV1-HP
                                                                                                                                         probes
                                                                              H2N3 A/Mallard/AB/77/1977                                                                                                                                                                                                                   H2N3 A/Mallard/AB/77/1977
                                                                              H3N2 A/TY/Eng/1969                                                                                                                                                                                                                          H3N2 A/TY/Eng/1969
                                                                              H4N8 A/Parrot/England/1970                                                                                                                                                                                                                  H4N8 A/Parrot/England/1970
                                                                              H5N1 A/CK/Scot/1959                                                                                                                                                                                                                         H5N1 A/CK/Scot/1959
                                                                              H5N2 A/CK/Hild/26654 1368/1994
                                                                                                     -                                                                                                                                                                                                                    H5N2 A/CK/Hild/26654 1368/1994
                                                                                                                                                                                                                                                                                                                                                 -
                                                                              H5N2 A/CK/Penn/1370                                                                                                                                                                                                                         H5N2 A/CK/Penn/1370
                                                                              H5N2 A/TK/Minn/3689 1551/1981
                                                                                                    -                                                                                                                                                                                                                     H5N2 A/TK/Minn/3689 1551/1981
                                                                                                                                                                                                                                                                                                                                                -
                                                                              H5N2 A/DK/BC/26-6/2000                                                                                                                                                                                                                      H5N2 A/DK/BC/26-6/2000
                                                                              H5N3 A/TY/CA/35621/1984                                                                                                                                                                                                                     H5N3 A/TY/CA/35621/1984




                                                                                                                  41 AIV strains
                                                                              H5N8 A/TY/Ireland/1963                                                                                                                                                                                                                      H5N8 A/TY/Ireland/1963




                                                                                                                                                                                                                                                                                                                                                              41 AIV strains
                                                                              H5N9 A/TY/ON/7732/1966                                                                                                                                                                                                                      H5N9 A/TY/ON/7732/1966
                                                                              H5N9 A/TY/Wisconsin/1968                                                                                                                                                                                                                    H5N9 A/TY/Wisconsin/1968
                                                                              H6N2 A/TY/Mass/1965                                                                                                                                                                                                                         H6N2 A/TY/Mass/1965
                                                                              H6N8 A/TY/ON/1963                                                                                                                                                                                                                           H6N8 A/TY/ON/1963
                                                                                                -
                                                                              H7N1 A/TY/ON/18 2/2000                                                                                                                                                                                                                                        -
                                                                                                                                                                                                                                                                                                                          H7N1 A/TY/ON/18 2/2000
                                                                              H7N3 A/CK/Aust/3634/1992                                                                                                                                                                                                                    H7N3 A/CK/Aust/3634/1992
                                                                              H7N3 A/CK/BC/514/2004                                                                                                                                                                                                                       H7N3 A/CK/BC/514/2004
                                                                              H7N7 A/CK/Victoria/32972/1985                                                                                                                                                                                                               H7N7 A/CK/Victoria/32972/1985
                                                                              H7N8 A/MR/China/28710/1995                                                                                                                                                                                                                  H7N8 A/MR/China/28710/1995
                                                                              H8N4 A/TY/ON/6118/1967                                                                                                                                                                                                                      H8N4 A/TY/ON/6118/1967
                                                                              H9N2 A/TY/Minn/12../1981                                                                                                                                                                                                                    H9N2 A/TY/Minn/12../1981
                                                                              H10N7 A/CK/Germany/1949                                                                                                                                                                                                                     H10N7 A/CK/Germany/1949
                                                                              H11N1 A/DK/Eng/1956                                                                                                                                                                                                                         H11N1 A/DK/Eng/1956
                                                                              H12N5 A/DK/AB/60/1976                                                                                                                                                                                                                       H12N5 A/DK/AB/60/1976
                                                                              H13N1 A/Gull/MD/704/1977                                                                                                                                                                                                                    H13N1 A/Gull/MD/704/1977
                                                                              H14N5 A/Mallard/Gurjev/263/1982                                                                                                                                                                                                             H14N5 A/Mallard/Gurjev/263/1982
                                                                              H15N8 A/DK/Aust/341/1983                                                                                                                                                                                                                    H15N8 A/DK/Aust/341/1983
                                                                              H16N3 A/BHG/Sweden/5/1999                                                                                                                                                                                                                   H16N3 A/BHG/Sweden/5/1999
                                                                              H2N9 A/Pintail/AB/293/7                                                                                                                                                                                                                     H2N9 A/Pintail/AB/293/7
                                                                              H4N6 A/DK/Czech/1956                                                                                                                                                                                                                        H4N6 A/DK/Czech/1956
                                                                              H9N9 A/Pheasant/WA/373                                                                                                                                                                                                                      H9N9 A/Pheasant/WA/373
                                                                              H3N8 A/Mallard/AB/431/1983                                                                                                                                                                                                                  H3N8 A/Mallard/AB/431/1983
                                                                              H5N2 A/Mallard/QC/2323 25/2006
                                                                                                       -                                                                                                                                                                                                                  H5N2 A/Mallard/QC/2323 25/2006
                                                                                                                                                                                                                                                                                                                                                   -
                                                                              H1N1 A/Mallard/AB/306/06                                                                                                                                                                                                                    H1N1 A/Mallard/AB/306/06
                                                                              H5N2 A/Goose/MB/428/2006                                                                                                                                                                                                                    H5N2 A/Goose/MB/428/2006
                                                                              H5N2 A/Mallard/BC/8038/2006




                                                                                                                                                                                                                                                                                                                                                              14 LP NDV strains 8 HP NDV strains
                                                                                                                                                                                                                                                                                                                          H5N2 A/Mallard/BC/8038/2006
                                                                              H10N7 A/CK/ON/01/1999                                                                                                                                                                                                                       H10N7 A/CK/ON/01/1999
                                                                              H11N1 A/Goose/MB/321/2006                                                                                                                                                                                                                   H11N1 A/Goose/MB/321/2006
                                                                              H13N6 A/Gull/SK/283/1999                                                                                                                                                                                                                    H13N6 A/Gull/SK/283/1999
                                                                              H1N1 A/Mallard/ON/269/2005                                                                                                                                                                                                                  H1N1 A/Mallard/ON/269/2005
                                                                              H1N2 A/Goose/MB/325/2006                                                                                                                                                                                                                    H1N2 A/Goose/MB/325/2006
                                                                              WIN-AH-2008-OTH-0027 276 KI ICE                                                                                                                                                                                                             WIN-AH-2008-OTH-0027 276 KI ICE
                                                                              NDV CK/US ROAKIN/48 2 CE                                                                                                                                                                                                                    NDV CK/US ROAKIN/48 2 CE
                                                                                                                        22 NDV strains




                                                                              00W1015 00.09.28 98 IMP 134 2CE                                                                                                                                                                                                             00W1015 00.09.28 98 IMP 134 2CE
                                                                              (NDV) DYHAMAZON ICE 99-10-25                                                                                                                                                                                                                (NDV) DYHAMAZON ICE 99-10-25
                                                                              PMV-1 Australia Velogenic d10                                                                                                                                                                                                               PMV-1 Australia Velogenic d10
                                                                              PMV-1 California Kimber Velogenic                                                                                                                                                                                                           PMV-1 California Kimber Velogenic
                                                                              PMV-1 Fontana 1083 Velogenic                                                                                                                                                                                                                PMV-1 Fontana 1083 Velogenic
                                                                              PMV-1 G.B. Texas Velogenic                                                                                                                                                                                                                  PMV-1 G.B. Texas Velogenic
                                                                              NDV B1                                                                                                                                                                                                                                      NDV B1
                                                                              PMV-1 Ireland/Ulster E3/76                                                                                                                                                                                                                  PMV-1 Ireland/Ulster E3/76
                                                                              PMV-1 LaSota Lentogenic                                                                                                                                                                                                                     PMV-1 LaSota Lentogenic
                                                                              PMV-1 Conneticut 1960                                                                                                                                                                                                                       PMV-1 Conneticut 1960
                                                                              WIN-AH-2008-OTH-0069                                                                                                                                                                                                                        WIN-AH-2008-OTH-0069
                                                                              WIN-AH-2003-CN-00013                                                                                                                                                                                                                        WIN-AH-2003-CN-00013
                                                                              WIN-AH-2003-CN-0009                                                                                                                                                                                                                         WIN-AH-2003-CN-0009
                                                                              WIN-AH-2001-NS-01416                                                                                                                                                                                                                        WIN-AH-2001-NS-01416
                                                                              WIN-AH-2008-FAV-0001                                                                                                                                                                                                                        WIN-AH-2008-FAV-0001
                                                                              WIN-AH-2005-NS-03042                                                                                                                                                                                                                        WIN-AH-2005-NS-03042
                                                                              WIN-AH-2006-IM-00028                                                                                                                                                                                                                        WIN-AH-2006-IM-00028
                                                                              WIN-AH-2006-NS-000924                                                                                                                                                                                                                       WIN-AH-2006-NS-000924
                                                                              WIN-AH-2001-CN-00017                                                                                                                                                                                                                        WIN-AH-2001-CN-00017
                                                                              96CNN0057                                                                                                                                                                                                                                   96CNN0057
                                                                              NTC                                                                                                                                                                                                                                         NTC
                                    Sensitivity of detection probes


           Sensitivity of detection probe to APMV-1 Am azon (HP)                      Sensitivity of detection probe to NDV B2 (LP)


      25                                                                         25
                                                                                                                                      10^5
                                                                   10^5
      20                                                                         20                                                   10^4
                                                                   10^4
                                                                                                                                      10^3
                                                                   10^3
      15                                                                         15                                                   10^2
                                                                   10^2




                                                                           P:N
P:N




                                                                                                                                      10^1
                                                                   10^1
      10                                                                         10                                                   10^0
                                                                   10^0
                                                                                                                                      10^-1
                                                                   10^-1
      5                                                                          5                                                    10^-2
                                                                   NTC
                                                                                                                                      NTC

      0                                                                          0
                  NDV-M-10                   FMD-AT-23                                    NDV-M-10                  FMD-AT-23

                                  Probe                                                                 Probe




       Limit of detection 103 TCID50 /ml                                     Limit of detection 101 TCID50/ml
                                  Sensitivity of Pathotyping Probes

                           Sensitivity of HP probe                                                     Sensitivity of LP probe


      14                                                                            14

      12                                                                            12

      10                                                                            10

      8      DYH Amazon (HP)                     B2 (LP)                             8          B2 (LP)                       DYH Amazon (HP)
P:N




                                                                              P:N
      6                                                                              6

      4                                                                              4
      2                                                                              2
      0                                                                              0
             HP 3            FMD-AT-23            HP3           FMD-AT-23                LP8 NC22         FMD-AT-23           LP8 NC22        FMD-AT-23
                                         Probe                                                                        Probe

           10^5     10^4   10^3   10^2    10^1   10^0   10^-1   10^-2   NTC              10^5   10^4   10^3   10^2     10^1    10^0   10^-1   10^-2   NTC




      Limit of detection 103 TCID50 / ml                                       Limit of detection 102 TCID50 / ml
                                          NDV Clinical Testing
Clinical samples                                                                                      Bird 642 Cloacal swab



10 birds infected with APMV-1 Amazon
                                                                              20
                                                                              18
                                                                              16

Sampling: Swabs: oropharynx, cloaca                                           14
                                                                              12
                                                                                                                                                  1dpi




                                                                        P:N
                                                                                                                                                  2 dpi
                                                                              10
                                                                                                                                                  3 dpi
                Dpi: 1, 2, 3                                                   8
                                                                               6
                                                                                                                                                  NTC

                                                                               4
Total = 60 samples                                                             2
                                                                               0
                                                                                       NDV-M-01            NDV-HP-3            FMD-AT-23
Field negative samples = 40                                                                                  Probe




                                                                         Comparison of Microarray to RT-PCR
                      Bird 642 Oropharyngeal swab                                                     Cloaca                    Oropharynx
      20
      18                                                                       DPI         Array            RT-PCR            Array        RT-PCR
      16
      14
                                                                1dpi
      12
P:N




      10
                                                                2 dpi
                                                                3 dpi
                                                                                   1              7              9              0            1
       8
                                                                NTC
       6
       4                                                                           2          10                10              5            8
       2
       0
           NDV-M-01           NDV-HP-3              FMD-AT-23                      3              7             10              8            10
                                 Probe
BSE facts
1997 Rum to rum feed ban – pet food. Livestock feedd, fetrilizers, skull,
brain, eyes tonsils, CFIA control collection
2001 National TSE network
2001 Canadian cattle ID
May 2003 National BSE case


TSE Lab Network scrapie associated fibrils

				
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