Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry
Abstract
1. Introduction
2. Materials and Methods
2.1. Reference Viruses and Field Samples Collection
2.2. Primer and Probe Design
2.3. Sensitivity Analyses of One-Step Multiplex RT-qPCR Analysis
2.4. One-Step Multiplex RT-qPCR Analysis
2.5. Specificity Analyses of One-Step Multiplex RT-qPCR Analysis
2.6. H5 HPAI Clade Assignment of Filed Samples
3. Results
3.1. Sensitivity of One-Step Multiplex RT-qPCR Assay
3.2. Specificity of One-Step Multiplex RT-qPCR Assay
3.3. Application of One-Step Multiplex RT-qPCR Assay Using Clinical Samples
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Specificity | Fluorescence Dye | Singleplex Assay | Multiplex Assay | ||||
---|---|---|---|---|---|---|---|
Limit of Detection | Mean ct Value | SD | Limit of Detection | Mean ct Value | SD | ||
Clade 2.3.4.4 | Hex | 5 × 101 | 34.39 | 0.025 | 5 × 101 | 33.84 | 0.03 |
Clade2.3.2.1 | FAM | 5 × 101 | 33.83 | 0.020 | 5 × 101 | 34.5 | 0.025 |
Pan-H5 HPAI | Texas red | 5 × 101 | 34.12 | 0.061 | 5 × 101 | 33.89 | 0.06 |
Virus Name | Host | Accession Number | Subtype | Mean ct Value | Comments | ||
---|---|---|---|---|---|---|---|
H5 HPAI | 2.3.2.1c Clade | 2.3.4.4 Clade | |||||
A/California/04/2009 | Human | GQ280797 | H1N1 | ND | ND | ND | |
A/wild bird/Korea/SK14/2014 | Avian | KX066871 | H1N1 | ND | ND | ND | LPAI |
A/swine/Korea/CAN04/2005 | Swine | EU798790 | H3N2 | ND | ND | ND | |
A/canine/Korea/01/2007 | Canine | JX163256 | H3N2 | ND | ND | ND | |
A/aquatic bird/SouthKorea/sw006/2016 | Avian | MG386182 | H3N3 | ND | ND | ND | LPAI |
A/equine/Kyonggi/SA1/2011 | Equine | JX844146 | H3N8 | ND | ND | ND | |
A/environment/Korea/W150/2006 | Avian | EU233739 | H5N1 | 21.55 | ND | ND | Clade 2.2 |
A/duck/Vietnam/NCVD-1648/2012 | Avian | KY171342 | H5N1 | 20.58 | 21.6 | ND | Clade 2.3.2.1c |
A/aquatic bird/Korea/CN2/2009 | Avian | KY584075 | H5N2 | ND | ND | ND | LPAI |
A/aquatic bird/ South Korea/sw007/2015 | Avian | MG386197 | H5N3 | ND | ND | ND | LPAI |
A/Environment/Korea/W541/2016 | Avian | KY272997 | H5N6 | 22.13 | ND | 21.36 | Clade 2.3.4.4 |
A/Common Teal/Korea/W555/2017 | Avian | KY576080 | H5N8 | 20.82 | ND | 19.02 | Clade 2.3.4.4 |
A/aquatic bird/ South Korea/sw001/2015 | Avian | MF987893 | H7N1 | ND | ND | ND | LPAI |
A/Chicken/Korea/MS96/1996 | Avian | GU053186 | H9N2 | ND | ND | ND | LPAI |
A/Aquatic bird/South Korea/SW1/2018 | Avian | MK539837 | H10N1 | ND | ND | ND | LPAI |
Infectious bronchitis virus | Avian | KY992863 | VNUA3 | ND | ND | ND | |
Newcastle disease virus | Avian | KC607878 | VN1 | ND | ND | ND | |
Coronavirus-229E, strain | Human | AY386391 | KUMC-9 | ND | ND | ND | |
Parainfluenza virus 1 | Human | MG255129 | KUMC-44 | ND | ND | ND |
Samples Description | ct Value | Clade ** | ||||||
---|---|---|---|---|---|---|---|---|
IAV * | HPAI | 2.3.2.1 | 2.3.4.4 | |||||
Singleplex | Multiplex | Singleplex | Multiplex | Singleplex | Multiplex | |||
A/environment/Korea/W150/2006 | + | 20.32 | 20.04 | ND | ND | ND | ND | 2.2 |
A/Environment/Korea/W541/2016 | + | 20.68 | 21.33 | ND | ND | 21.32 | 22.09 | 2.3.4.4 |
A/Common Teal/Korea/W555/2017 | + | 19.79 | 21.81 | ND | ND | 21.62 | 22.01 | 2.3.4.4 |
Feces sample #1 | + | 25.35 | 26.3 | ND | ND | ND | ND | 1 |
Feces sample #2 | + | 21.82 | 21.29 | 23.72 | 24.07 | ND | ND | 2.3.2.1a |
Feces sample #3 | + | 19.5 | 19.32 | 20.97 | 22.12 | ND | ND | 2.3.2.1a |
Feces sample #4 | + | 19.84 | 19.72 | 20.05 | 20.93 | ND | ND | 2.3.2.1c |
Feces sample #5 | + | 19.99 | 19.2 | 19.89 | 21.32 | ND | ND | 2.3.2.1c |
Feces sample #6 | + | 21.37 | 21.53 | 22.18 | 21.9 | ND | ND | 2.3.2.1c |
Feces sample #7 | + | 20.24 | 20.35 | 21.07 | 20.75 | ND | ND | 2.3.2.1c |
Feces sample #8 | + | 19.88 | 19.57 | 19.93 | 20.49 | ND | ND | 2.3.2.1c |
Feces sample #9 | + | 23.13 | 22.63 | 23.19 | 23.56 | ND | ND | 2.3.2.1c |
Feces sample #10 | + | 22.29 | 22.15 | 22.64 | 23.26 | ND | ND | 2.3.2.1c |
Feces sample #11 | + | 20.33 | 19.68 | 20.4 | 21.1 | ND | ND | 2.3.2.1c |
Feces sample #12 | + | 20.13 | 21 | ND | ND | 20.7 | 21.97 | 2.3.4.4 |
Feces sample #13 | + | 19.48 | 22.59 | ND | ND | 22.33 | 21.24 | 2.3.4.4 |
Feces sample #14 | + | 20.2 | 19.97 | 20.54 | 20.45 | ND | ND | 2.3.2.1c |
Feces sample #15 | + | 22.05 | 21.58 | 21.93 | 22.31 | ND | ND | 2.3.2.1c |
Feces sample #16 | + | 21.64 | 23.66 | ND | ND | 23.54 | 23.45 | 2.3.4.4 |
Feces sample #17 | + | 20.85 | 20.56 | 20.88 | 21.44 | ND | ND | 2.3.2.1c |
Feces sample #18 | + | 19.49 | 19.65 | 20.15 | 20.78 | ND | ND | 2.3.2.1c |
Feces sample #19 | + | 20.92 | 21.02 | 21.6 | 21.98 | ND | ND | 2.3.2.1c |
Feces sample #20 | + | 20.68 | 20.31 | 20.87 | 21.26 | ND | ND | 2.3.2.1c |
Feces sample #21 | + | 22.04 | 23.54 | ND | ND | 23.15 | 23.13 | 2.3.4.4 |
Feces sample #22 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #23 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #24 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #25 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #26 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #27 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #28 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #29 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #30 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #31 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #32 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #33 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #34 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #35 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #36 | + | 23.21 | 24.33 | ND | ND | 23.86 | 23.92 | 2.3.4.4 |
Feces sample #37 | + | 26.71 | 27.4 | 27.01 | 27.48 | ND | ND | 2.3.2.1 |
Feces sample #38 | + | 20.49 | 21.48 | 21.01 | 20.98 | ND | ND | 2.3.2.1c |
Feces sample #39 | + | 22.27 | 22.93 | 22.65 | 23.11 | ND | ND | 2.3.2.1c |
Feces sample #40 | + | 21.89 | 22.36 | 21.82 | 22.56 | ND | ND | 2.3.2.1c |
Feces sample #41 | + | 22.92 | 23.31 | 22.91 | 23.37 | ND | ND | 2.3.2.1c |
Feces sample #42 | + | 19.46 | 19.62 | 19.38 | 19.96 | ND | ND | 2.3.2.1a |
Feces sample #43 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #44 | + | 23.17 | 23.71 | 23.16 | 24.16 | ND | ND | 2.3.2.1c |
Feces sample #45 | + | 21.62 | 22.37 | 21.88 | 22.75 | ND | ND | 2.3.2.1c |
Feces sample #46 | + | 29.58 | 29.98 | 29.41 | 30.42 | ND | ND | 2.3.2.1c |
Feces sample #47 | + | 23.36 | 23.21 | 23.86 | 23.76 | ND | ND | 2.3.2.1c |
Feces sample #48 | + | 18.88 | 18.94 | ND | ND | 19.88 | 20.05 | 2.3.4.4 |
Feces sample #49 | + | 23.6 | 24.3 | 23.42 | 24.52 | ND | ND | 2.3.2.1c |
Feces sample #50 | + | 23.9 | 24.14 | 24.16 | 24.6 | ND | ND | 2.3.2.1c |
Feces sample #51 | + | 22.32 | 22.85 | 22.71 | 23.22 | ND | ND | 2.3.2.1c |
Feces sample #52 | + | 24.67 | 24.4 | 24.36 | 24.37 | ND | ND | 2.3.2.1c |
Feces sample #53 | + | 21.32 | 22.06 | 21.22 | 21.71 | ND | ND | 2.3.2.1c |
Feces sample #54 | + | 18.97 | 18.53 | ND | ND | 18.3 | 18.94 | 2.3.4.4 |
Feces sample #55 | + | 23.3 | 23.22 | 23.17 | 22.93 | ND | ND | 2.3.2.1c |
Feces sample #56 | + | 22.42 | 22.79 | 22.94 | 23 | ND | ND | 2.3.2.1c |
Feces sample #57 | + | 22.8 | 22.92 | 22.43 | 22.36 | ND | ND | 2.3.2.1c |
Feces sample #58 | + | 25.22 | 25.04 | 24.17 | 25.05 | ND | ND | 2.3.2.1c |
Feces sample #59 | + | 23.57 | 23.83 | 23.91 | 23.38 | ND | ND | 2.3.2.1c |
Feces sample #60 | + | 23.06 | 23.78 | 23.54 | 23.39 | ND | ND | 2.3.2.1c |
Feces sample #61 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #62 | + | 24.81 | 24.75 | 23.93 | 24.55 | ND | ND | 2.3.2.1c |
Feces sample #63 | + | 18.6 | 18.09 | ND | ND | 18.93 | 19.16 | 2.3.4.4 |
Feces sample #64 | + | 18.16 | 18.2 | ND | ND | 19.16 | 19.3 | 2.3.4.4 |
Feces sample #65 | + | ND | ND | ND | ND | ND | ND | LPAI |
Feces sample #66 | + | 23.79 | 23.94 | 24.28 | 24.4 | ND | ND | 2.3.2.1a |
Feces sample #67 | + | 23.78 | 23.78 | 23.83 | 24.25 | ND | ND | 2.3.2.1a |
Feces sample #68 | + | 24.25 | 24.37 | 24.47 | 24.66 | ND | ND | 2.3.2.1c |
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Le, T.B.; Kim, H.K.; Na, W.; Le, V.P.; Song, M.-S.; Song, D.; Jeong, D.G.; Yoon, S.-W. Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry. Viruses 2020, 12, 100. https://doi.org/10.3390/v12010100
Le TB, Kim HK, Na W, Le VP, Song M-S, Song D, Jeong DG, Yoon S-W. Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry. Viruses. 2020; 12(1):100. https://doi.org/10.3390/v12010100
Chicago/Turabian StyleLe, Tran Bac, Hye Kwon Kim, Woonsung Na, Van Phan Le, Min-Suk Song, Daesub Song, Dae Gwin Jeong, and Sun-Woo Yoon. 2020. "Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry" Viruses 12, no. 1: 100. https://doi.org/10.3390/v12010100
APA StyleLe, T. B., Kim, H. K., Na, W., Le, V. P., Song, M.-S., Song, D., Jeong, D. G., & Yoon, S.-W. (2020). Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry. Viruses, 12(1), 100. https://doi.org/10.3390/v12010100