An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Samples and Nucleic Acid Extraction
2.2. Reference qRT-PCR Assays
2.3. Reference Gene Construction for dqRT-PCR Assay
2.4. Primers and Probe for dqRT-PCR Assay
2.5. Optimization of dqRT-PCR Conditions
2.6. Sensitivity of dqRT-PCR Assays
2.7. Specificity of dqRT-PCR Assays
2.8. Precision of dqRT-PCR Assays
2.9. Clinical Evaluation of dqRT-PCR Assay
3. Results
3.1. Interpretation of the dqRT-PCR Assay
3.2. Specificity and Sensitivity of the dqRT-PCR Assay
3.3. Precision of the dqRT-PCR Assay
3.4. Comparative Clinical Evaluation of the dqRT-PCR Assay
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Method | Target Gene | Primer and Probe | Primer/Probe Sequence (5′–3′) a | Genome Position b | Amplicon Size (bp) | Reference |
---|---|---|---|---|---|---|
dqRT-PCR | L | CPIV-LF | GCTTGGACAATGATATCTATCTC | 10649–10671 | 83 | This study |
CPIV-LR | TCTCCCTGCACCATACTC | 10714–10731 | ||||
CPIV-LP | FAM–CCACAGAGTCTGGCACACGAGTA–BHQ1 | 10689–10711 | ||||
Canine 16S rRNA | EIPC-F | AGACGAGAAGACCCTATG | 2142–2159 | 116 | This study | |
EIPC-R | GGTCACCCCAACCTAAAT | 2240–2257 | ||||
EIPC-P | HEX–ACCTACAAGGCATAACATAACACCA–BHQ1 | 2199–2223 | ||||
qRT-PCR | HN | cPIV-1278f | GCCATCTGCTATATCAGCTCAGAA | 7861–7884 | 101 | Windsor et al. [28] |
cPIV-1378r | TCAAGCAAAAACCGGGACAT | 7942–7961 | ||||
cPIV-1320p | FAM–AGCAGCCTCCTGTCCCAGGTCTAGGG–BHQ1 | 7903–7928 | ||||
N | NF | GATCATTCCGCTTAATCCCC | 480–499 | 77 | Dong et al. [27] | |
NR | TTCTGCAAGTGCAGCATAGG | 537–556 | ||||
NP | FAM–TCGTTCAGGTATGAGCCGTGGA–BHQ1 | 505–526 |
Pathogen | Strain | Source a | Amplification of Target Gene | |
---|---|---|---|---|
CPIV5 (FAM) | EIPC (HEX) | |||
Canine parainfluenza virus 5 | D008 | CAVS | + | + |
Canine parainfluenza virus 5 | Cornell | CAVS | + | + |
Canine parainfluenza virus 5 | NL-CPI-5 | CAVS | + | + |
Canine parainfluenza virus 5 | Korean isolate | ADIC | + | + |
Canine respiratory coronavirus | K37 | APQA | − | − |
Canine coronavirus | K378 | CAVS | − | − |
Canine distemper virus | Onderstepoort | CAVS | − | + |
Canine influenza virus | A/Canine/Republic of Korea/01/07(H3N2) | CAVS | − | + |
Canine adenovirus 2 | Ditchfield | CAVS | − | + |
Canine parvovirus | 7809 16-LP | CAVS | − | + |
Bordetella bronchiseptica | S-55 | CAVS | − | + |
Non-infected canine swab sample | - | APQA | − | + |
Non-infected canine lung sample | - | APQA | − | + |
MDCK cell | - | ADIC | − | + |
ST cell | - | ADIC | − | − |
Vero cell | - | ADIC | − | − |
Dilutions (TCID50/mL) | Results of Different Assays a | ||||||||
---|---|---|---|---|---|---|---|---|---|
L gene-Specific dqRT-PCR | HN Gene-Specific qRT-PCR | N Gene-Specific qRT-PCR | |||||||
1-Folds | 2-Folds | 3-Folds | 1-Folds | 2-Folds | 3-Folds | 1-Folds | 2-Folds | 3-Folds | |
104 | 21.49 | 21.53 | 21.52 | 22.10 | 22.74 | 22.49 | 21.79 | 21.87 | 21.86 |
103 | 24.75 | 24.97 | 24.94 | 25.82 | 26.08 | 26.19 | 25.14 | 25.37 | 25.29 |
102 | 28.03 | 28.10 | 28.16 | 29.59 | 29.85 | 30.03 | 28.50 | 28.28 | 28.35 |
101 | 31.26 | 31.46 | 34.35 | 32.99 | 33.53 | 33.17 | 31.39 | 31.33 | 31.47 |
100 | 34.95 | 33.97 | 34.11 | 37.75 | NC | 36.85 | 34.94 | 34.32 | 34.45 |
10−1 | NC | NC | NC | NC | NC | NC | NC | NC | NC |
Concentration of RNA (Copies/Reaction) | Ct Values of the dqRT-PCR for CPIV5 RNAs | |||||
---|---|---|---|---|---|---|
Intra-Assay | Inter-Assay | |||||
Mean | SD | CV (%) | Mean | SD | CV (%) | |
High (106) | 20.73 | 0.03 | 0.1 | 20.33 | 0.29 | 1.4 |
Medium (104) | 27.34 | 0.11 | 0.4 | 26.60 | 0.51 | 1.9 |
Low (102) | 34.45 | 0.42 | 1.2 | 33.40 | 0.66 | 2.0 |
Test Results of Different Assays | New dqRT-PCR | Detection Rate | Overall Agreement | |||
---|---|---|---|---|---|---|
Positive | Negative | Total | ||||
HN gene-specific qRT-PCR | Positive | 15 | 0 | 15 | 5.6% | 99.6% |
Negative | 1 | 253 | 254 | |||
Total | 16 | 253 | 269 | |||
N gene-specific qRT-PCR | Positive | 16 | 0 | 16 | 5.9% | 100.0% |
Negative | 0 | 253 | 253 | |||
Total | 16 | 253 | 269 | |||
Detection rate | 5.9% |
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Jeon, G.-T.; Kim, H.-R.; Shin, Y.-K.; Kwon, O.-K.; Kang, H.-E.; Kwon, O.-D.; Park, C.-K. An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5. Vet. Sci. 2023, 10, 142. https://doi.org/10.3390/vetsci10020142
Jeon G-T, Kim H-R, Shin Y-K, Kwon O-K, Kang H-E, Kwon O-D, Park C-K. An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5. Veterinary Sciences. 2023; 10(2):142. https://doi.org/10.3390/vetsci10020142
Chicago/Turabian StyleJeon, Gyu-Tae, Hye-Ryung Kim, Yeun-Kyung Shin, Oh-Kyu Kwon, Hae-Eun Kang, Oh-Deog Kwon, and Choi-Kyu Park. 2023. "An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5" Veterinary Sciences 10, no. 2: 142. https://doi.org/10.3390/vetsci10020142
APA StyleJeon, G. -T., Kim, H. -R., Shin, Y. -K., Kwon, O. -K., Kang, H. -E., Kwon, O. -D., & Park, C. -K. (2023). An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5. Veterinary Sciences, 10(2), 142. https://doi.org/10.3390/vetsci10020142