Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Examined Samples
2.2. DNA Extraction
2.3. Conventional Polymerase Chain Reaction and Nested Polymerase Chain Reaction
2.4. Cloning and Sequence Analysis of the Amplicons
2.5. Real-Time Polymerase Chain Reaction
2.6. Software
3. Results
3.1. Characterization of DNA from Samples Collected by Non-Invasive and Low-Invasive Methods
3.2. Comparative Analysis of DNA Samples from Aachen Minipigs by Nested Polymerase Chain Reaction
3.3. Porcine Cytomegalovirus Sequences Detected in Piglets
3.4. Detection of Porcine Cytomegalovirus by Real-Time Polymerase Chain Reaction and Comparison of the Sensitivity of Uniplex and Duplex Systems
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Primers (nt) | Sequence (5′-3′) | Tm (°C) | Oligonucleotide Length | Homo-Dimers. ΔG below −7 kcal/mol | Tm Hairpin (°C) |
---|---|---|---|---|---|
Conventional Polymerase Chain Reaction (PCR) | |||||
F1 (63–81) * | ACGGGGATCGACGAGAAAG | 66.4 | 19 | none | 49 |
R1 (412–390) * | CTAGACGAGAGGACATTGTTGAT | 61.1 | 23 | none | 29 |
F2 (182–201)* | GAAGAGAAAGGAAGTGAAGG | 57.1 | 20 | none | None |
R2 (386–368) * | GTCACTCGTCTGCCTAAGC | 60.2 | 19 | none | 30 |
Real-time PCR | |||||
Fr-t (279–299) * | AATGCGTTTTACAACTTCACG | 61.5 | 21 | none | 29 |
Rr-t (373–354) * | CTGAGCATGTCCCGCCCTAT | 67.4 | 20 | none | 19 |
Probe (331–350) * | 6FAM-CTCTAGCGGCGTCCATCACC-BHQ | 69.2 | 20 | none | 17 |
F cyclophilin A (174–196) ** | TGCTTTCACAGAATAATTCCAGGATTTA | 59.1 | 28 | none | 23 |
R cyclophilin A (250–230) ** | GACTTGCCACCAGTGCCATTA | 61.7 | 21 | none | 22 |
Probe cyclophilin A (205–228) ** | Cy5-TGCCAGGGTGGTGACTTCACACGCC-BHQ | 67.1 | 25 | none | 44 |
Piglet #/Material | Sera | Anal Swabs | Ears Biopsies | Oral Swabs |
---|---|---|---|---|
1 | 29.7 | 22.2 | 22.3 | 26.1 |
2 | 29.6 | 23.4 | 24.6 | 24.3 |
3 | 27.8 | 21.5 | 23.0 | 25.5 |
4 | 26.8 | 21.8 | 22.0 | 25.5 |
5 | 27.7 | 21.6 | 20.5 | 28.5 |
6 | 27.8 | 21.7 | 20.7 | 26.4 |
7 | 28.3 | 21.9 | 21.3 | 28.5 |
8 | 28.3 | 22.3 | 23.5 | 28.0 |
9 | 28.2 | 25.6 | 21.6 | 26.7 |
10 | 27.1 | 23.6 | 21.0 | 25.9 |
Mean values: | 28.13 | 22.56 | 22.05 | 26.54 |
Copies/reaction: | 6 × 103 | 5 × 106 | 5 × 106 | 2 × 104 |
Copy/µg: | 1.2 × 105 | 2.5 × 107 | 2.5 × 107 | 1 × 105 |
Piglet # | Sera Uniplex Duplex | Oral Swabs Uniplex Duplex | Ears Biopsies Uniplex Duplex | Anal Swabs Uniplex Duplex | ||||
---|---|---|---|---|---|---|---|---|
1 | Neg. | Neg. | 30.0 | 30.0 | 35.8 | Neg. | Neg. | Neg. |
2 | Neg. | Neg. | 27.5 | 28.2 | 30.9 | 31.8 | 39.7 | Neg. |
3 | 38.6 | Neg. | 29.2 | 30.9 | Neg. | Neg. | Neg. | Neg. |
4 | 36.6 | Neg. | 28.4 | 30.8 | 39.4 | Neg. | 37.0 | Neg. |
5 | Neg. | Neg. | 27.3 | 29.1 | 34.0 | 41.3 | 36.2 | Neg. |
6 | 37.1 | Neg. | 28.1 | 28.6 | Neg. | Neg. | 41.3 | Neg. |
7 | Neg. | Neg. | 30.1 | 31.1 | Neg. | Neg. | Neg. | Neg. |
8 | 40.1 | Neg. | 29.4 | 31.4 | 32.3 | 39.7 | 39.5 | Neg. |
9 | 35.2 | Neg. | 26.1 | 26.9 | 35.2 | Neg. | 42.0 | Neg. |
10 | 37.2 | Neg. | 30.6 | Neg. | 43.0 | Neg. | 40.4 | Neg. |
Piglet # | Sample | DNA Load (ng) | PCR | Nested PCR | Uniplex Real-Time PCR (Ct Values) | G.e./Reaction |
---|---|---|---|---|---|---|
1 | Serum | 50 | Neg. | Neg. | Neg. | Neg. |
Anal swab | 200 | Neg. | Neg. | Neg. | Neg. | |
Oral swab | 200 | + | + | 30.0 | ~103 | |
Ear biopsies | 200 | Neg. | + | 35.8 | ~40 | |
2 | Serum | 50 | Neg. | Neg. | Neg. | Neg. |
Anal swab | 200 | Neg. | + | 39.7 | ~2–5 | |
Oral swab | 200 | + | + | 27.5 | 104 | |
Ear biopsies | 200 | Neg. | + | 30.9 | ~103 | |
3 | Serum | 50 | Neg. | Neg. | 38.6 | ~10 |
Anal swab | 200 | Neg. | Neg. | Neg. | Neg. | |
Oral swab | 200 | + | + | 29.2 | ~2 × 103 | |
Ear biopsies | 200 | Neg. | Neg. | Neg. | Neg. | |
4 | Serum | 50 | + | + | 36.6 | ~20 |
Anal swab | 200 | Neg. | + | 36.9 | ~20 | |
Oral swab | 200 | + | + | 28.4 | ~7 × 103 | |
Ear biopsies | 200 | Neg. | + | 39.4 | ~2–5 | |
5 | Serum | 50 | Neg. | Neg. | Neg. | Neg. |
Anal swab | 200 | Neg. | Neg. | 36.2 | ~20 | |
Oral swab | 200 | + | + | 27.3 | ~104 | |
Ear biopsies | 200 | Neg. | + | 34.0 | 10e2 | |
6 | Serum | 50 | Neg. | Neg. | 37.1 | 10 |
Anal swab | 200 | Neg. | Neg. | 41.3 | ~1–2 | |
Oral swab | 200 | + | + | 28.1 | ~7 × 103 | |
Ear biopsies | 200 | Neg. | Neg. | Neg. | Neg. | |
7 | Serum | 50 | Neg. | Neg. | Neg. | Neg. |
Anal swab | 200 | Neg. | Neg. | Neg. | Neg. | |
Oral swab | 200 | + | + | 30.1 | 103 | |
Ear biopsies | 200 | Neg. | Neg. | Neg. | Neg. | |
8 | Serum | 50 | Neg. | Neg. | 40.1 | ~2 |
Anal swab | 200 | Neg. | +(weak) | 39.5 | ~2 | |
Oral swab | 200 | + | + | 30.4 | 103 | |
Ear biopsies | 200 | Neg. | Neg. | 40.1 | ~2 | |
9 | Serum | 50 | Neg. | Neg. | 35.2 | ~30 |
Anal swab | 200 | Neg. | Neg. | 41.9 | ~1–2 | |
Oral swab | 200 | + | + | 26.1 | 2 × 104 | |
Ear biopsies | 200 | Neg. | Neg. | 35.2 | ~40 | |
10 | Serum | 50 | Neg. | Neg. | 37.2 | ~10 |
Anal swab | 200 | Neg. | Neg. | 40.4 | ~2 | |
Oral swab | 200 | + | + | 30.6 | 103 | |
Ear biopsies | 200 | Neg. | Neg. | 38.1 | ~5 |
Samples/Methods | PCR (Positive/Total) | Nested PCR (Positive/Total) | Uniplex Real-Time PCR (Positive/Total) | Duplex Real-Time PCR (Positive/Total) | G.e., Detected |
---|---|---|---|---|---|
Sera | 1/10 | 1/10 | 6/10 | 0/10 | 10–150/mL |
Anal swabs | 0/10 | 3/10 | 7/10 | 0/10 | 5–100/μg |
Oral swabs | 10/10 | 10/10 | 10/10 | 9/10 | 5 × 103–1 × 105/μg |
Ear biopsies | 0/10 | 1/10 | 7/10 | 3/10 | 25–5 × 103/μg |
Total | 11/40 | 15/40 | 30/40 | 12/40 |
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Morozov, V.A.; Heinrichs, G.; Denner, J. Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets. Viruses 2017, 9, 9. https://doi.org/10.3390/v9010009
Morozov VA, Heinrichs G, Denner J. Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets. Viruses. 2017; 9(1):9. https://doi.org/10.3390/v9010009
Chicago/Turabian StyleMorozov, Vladimir A., Gerd Heinrichs, and Joachim Denner. 2017. "Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets" Viruses 9, no. 1: 9. https://doi.org/10.3390/v9010009