A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Control Sample from Vaccines
2.2. Field Samples
2.3. DNA Extraction and Purification
2.4. Multiplex Primer Design
2.5. Evaluation of Primers
2.6. Optimizing Multiplex PCR
2.7. Determining the Limit of Detection of the Multiplex PCR
2.8. Detection of ILTV and ORT in Field Samples by Multiplex PCR Assay
3. Results
3.1. Optimization of Multiplex PCR
3.2. Specificity and Limit of Detection of Multiplex PCR
3.3. Application of Multiplex PCR on Field Samples
4. Discussion
4.1. Selection of the Target Pathogens and Detection Method
4.2. Multiplex Primer Design and Validation
4.3. Application of the Newly Developed Multiplex PCR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Forward/Reverse 1 | Gene | Sequence (5′-3′) | Reference |
---|---|---|---|
TK_792F | TK | TGGCAACGGATTCCGGCA | [29] |
TK_673R | TTGCAAATAGCGTCTGGTCGATTGAA | [43] | |
Or01_1607F | Or01 | GCTGAAATTATAAAAATGCC | [17] |
Or01_1607R | CACAAGCATAGACATTGG | [17] | |
Or01_1315F | ACATCAGCGGTTTGGTAAGC | This study | |
Or01_1315R | AGTACTTCCAGTTGCTCCGT | This study |
Forward/Reverse 1 | Pathogen | Sequence (5′-3′) | Expected Size (bp) | Reference |
---|---|---|---|---|
Pox.Po681R | FWPV | CGCCGCATCATCTACTTATC | 681 | [56] |
Pox.Po681F | CCACACAGCGCCATTCATTA | |||
TspE4C2.152F | E. coli | CACTATTCGTAAGGTCATCC | 152 | [57] |
TspE4C2.152R | AGTTTATCGCTGCGGGTCGC | |||
PM.460F | PM | GCTGTAAACGAACTCGCCAC | 460 | [58] |
PM.460R | ATCCGCTATTTACCCAGTGG | |||
MS.207F | MS | GAGAAGCAAAATAGTGATATCA | 202 | [59] |
MS.207R | CAGTCGTCTCCGAAGTTAACAA | |||
MG.732F | MG | GGATCCCATCTCGACCACGAGAAAA | 732 | [60] |
MG.732R | CTTTCAATCAGTGAGTAACTGATGA | |||
APG.500F | APG | CAATGTCGATCCTGGTACAATGAG | 500 | [61] |
APG.500R | CAAGGTATCGATCGTCTCTCTACT |
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Forward/Reverse | Pathogen | Sequence (5′-3′) | Expected Size (bp) |
---|---|---|---|
5ILTV.233F | ILTV | CCGACTTTCGCCGCGTTGTACT | 233 |
/5ILTV.233R | ACGAGACGCCTCCCGACATTCA | ||
6ORT.467F | ORT | AGCGGTGGAGGTGCTAGCCAAT | 467 |
/6ORT.467R | GCAGCAGGCGCTGGAGTTTCTT |
Experiment | Template DNA | Primer Mix | Expected Result | ||
---|---|---|---|---|---|
Non-Target 1 | Target 2 | ILTV | ORT | ||
1 | None | ILTV | 5ILTV.233F /5ILTV.233R and 6ORT.467F /6ORT.467R | + | - |
2 | None | ORT | - | + | |
3 | None | ILTV; ORT. | + | + | |
4 | None | Water | - | - | |
5 | Single | None | - | - | |
6 | Single | ILTV | + | - | |
7 | Single | ORT | - | + | |
8 | Single | ILTV; ORT. | + | + | |
9 | Double | ILTV | + | - | |
10 | Double | ORT | - | + | |
11 | Double | ILTV; ORT. | + | + | |
12 | Triple | ILTV | + | - | |
13 | Triple | ORT | - | + | |
14 | Triple | ILTV; ORT. | + | + |
5ILTV.233F/5ILTV.233R Mix (pmol/µL) | |||||
---|---|---|---|---|---|
(10) | (5) | (2.5) | (1.25) | ||
6ORT.467F/6ORT.467R Mix (pmol/µL) | (10) | 1 | 2 | 3 | 4 |
(5) | 5 | 6 | 7 | 8 | |
(2.5) | 9 | 10 | 11 | 12 | |
(1.25) | 13 | 14 | 15 | 16 |
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Nguyen, V.-G.; Cao, T.-B.-P.; Le, V.-T.; Truong, H.-T.; Chu, T.-T.-H.; Dang, H.-A.; Nguyen, T.-H.; Le, T.-L.; Huynh, T.-M.-L. A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale. Vet. Sci. 2023, 10, 272. https://doi.org/10.3390/vetsci10040272
Nguyen V-G, Cao T-B-P, Le V-T, Truong H-T, Chu T-T-H, Dang H-A, Nguyen T-H, Le T-L, Huynh T-M-L. A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale. Veterinary Sciences. 2023; 10(4):272. https://doi.org/10.3390/vetsci10040272
Chicago/Turabian StyleNguyen, Van-Giap, Thi-Bich-Phuong Cao, Van-Truong Le, Ha-Thai Truong, Thi-Thanh-Huong Chu, Huu-Anh Dang, Thi-Hoa Nguyen, Thi-Luyen Le, and Thi-My-Le Huynh. 2023. "A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale" Veterinary Sciences 10, no. 4: 272. https://doi.org/10.3390/vetsci10040272