Direct Detection of Feline Coronavirus by Three Rapid Antigen Immunochromatographic Tests and by Real-Time PCR in Cat Shelters
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Sampling
2.2. RNA Isolation and Reverse Transcription
2.3. Real-Time PCR—Identification of FCoV
2.4. RIM Tests
- taking a small amount of faecal sample (in the case of tests A and C, the disposable swab was inserted into faeces to take the required amount; in the case of test B, a small plastic applicator was used to take a sufficient amount of sample),
- inserting and mixing the faecal sample with assay diluent in the diluent tube,
- placing the tube with the sample on a flat horizontal surface for sedimentation of gross faecal particles,
- application of the supernatant on the test device (applying a few drops of supernatant into the sample well of a cassette in the case of tests A and B; placing the dipstick vertically into the sample tube in the case of RIM test C),
- reading the test result visually—a correct test procedure was indicated by the appearance of a control line; if the line did not appear, the result was considered invalid; the appearance of a test line and a control line indicated the presence of FCoV antigen, if only a control line appeared in the result window, no FCoV antigen was present in the sample.
2.5. Determination of Sensitivity and Specificity of RIM Tests
2.6. Statistical Analysis
3. Results
3.1. Real-Time PCR Analysis, Faecal Consistency
3.2. RIM Tests
3.3. Comparison of Results Given by RIM Tests and by Real-Time PCR
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Age Category | Classification of Result | |
---|---|---|
Positive (%) | Negative (%) | |
≤6 months | 6 (60) | 4 (40) |
6 < x ≤ 12 months | 7 (43.8) | 9 (56.2) |
1 < x ≤ 8 years | 27 (71.1) | 11 (28.9) |
x > 8 years | 4 (66.7) | 2 (33.3) |
Sex | Classification of Result | |
---|---|---|
Positive (%) | Negative (%) | |
female | 23 (67.6) | 11 (32.4) |
male | 23 (63.9) | 13 (36.1) |
Faecal Consistency Score | Classification of Result | |
---|---|---|
Positive (%) | Negative (%) | |
1—firm, well-formed stool | 22 (62.9) | 13 (37.1) |
2—mostly formed but a softer stool | 15 (68.2) | 7 (31.8) |
3—mostly unformed, watery stool | 7 (53.8) | 6 (46.2) |
Antibody | Sensitivity Stated by the Manufacturer | Specificity Stated by the Manufacturer | Kit Storage | Sample Storage | The Total Duration of the Test Procedure | Number of Invalid Attempts (a New Test Device Had to Be Used) | The Test Requires the Use of Additional Equipment That Is Not Included in the Kit | |
---|---|---|---|---|---|---|---|---|
RIM test A | anti-FCoV monoclonal antibody | >95% vs. PCR | >95% vs. PCR | 15–30 °C | fresh faeces; 2–8 °C for max. 24 h; longer storage at −20 °C or below | approx. 15 min | 1 | no |
RIM test B | anti-FCoV monoclonal antibody | >95% vs. PCR | >95% vs. PCR | 15–25 °C | fresh faeces; 2–8 °C for max. 4 days; longer storage at −20 °C or below | approx. 20 min | 3 | no |
RIM test C | anti-FCoV monoclonal antibody | >90% vs. ELISA, % vs. PCR unknown | >95% vs. ELISA, % vs. PCR unknown | 2–30 °C | fresh faeces; 2–8 °C for max. 24 h; longer storage at −20 °C or below | approx. 15 min | 6 | Yes (use of centrifuge is needed) |
RIM Test | Classification of Result | |
---|---|---|
Positive (%) | Negative (%) | |
RIM test A | 9 (12.9) | 61 (87.1) |
RIM test B | 36 (51.42) | 34 (48.6) |
RIM test C | 17 (24.3) | 53 (75.7) |
RIM Test | Faecal Consistency Score | |
---|---|---|
Spearman’s Rank Correlation Coefficient | p-Value | |
RIM test A | −0.1662 | 0.1692 |
RIM test B | −0.1878 | 0.1195 |
RIM test C | −0.1964 | 0.1033 |
RIM Test A (%) | RIM Test B (%) | RIM Test C (%) | |
---|---|---|---|
the total number of samples identified correctly (out of 70) | 35 (50.0) | 48 (68.6) | 39 (55.7) |
number of samples correctly identified as positive | 9 (12.9) | 29 (41.4) | 15 (21.4) |
number of samples correctly identified as negative | 26 (37.1) | 19 (27.2) | 24 (34.3) |
the total number of samples identified incorrectly (out of 70) | 35 (50.0) | 22 (31.4) | 31 (44.3) |
number of samples incorrectly identified as positive–false positive samples | 0 (0) | 7 (10.0) | 2 (2.9) |
number of samples incorrectly identified as negative–false negative samples | 35 (50.0) | 15 (21.4) | 29 (41.4) |
sensitivity | 20.5 | 65.9 | 34.1 |
specificity | 100 | 73.1 | 92.3 |
RIM Test | Number of Virus Particles | |
---|---|---|
Spearman’s Rank Correlation Coefficient | p-Value | |
RIM test A | 0.2334 | 0.0536 |
RIM test B | 0.2105 | 0.0825 |
RIM test C | 0.1773 | 0.1450 |
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Vojtkovská, V.; Lukešová, G.; Voslářová, E.; Konvalinová, J.; Večerek, V.; Lobová, D. Direct Detection of Feline Coronavirus by Three Rapid Antigen Immunochromatographic Tests and by Real-Time PCR in Cat Shelters. Vet. Sci. 2022, 9, 35. https://doi.org/10.3390/vetsci9020035
Vojtkovská V, Lukešová G, Voslářová E, Konvalinová J, Večerek V, Lobová D. Direct Detection of Feline Coronavirus by Three Rapid Antigen Immunochromatographic Tests and by Real-Time PCR in Cat Shelters. Veterinary Sciences. 2022; 9(2):35. https://doi.org/10.3390/vetsci9020035
Chicago/Turabian StyleVojtkovská, Veronika, Gabriela Lukešová, Eva Voslářová, Jarmila Konvalinová, Vladimír Večerek, and Dana Lobová. 2022. "Direct Detection of Feline Coronavirus by Three Rapid Antigen Immunochromatographic Tests and by Real-Time PCR in Cat Shelters" Veterinary Sciences 9, no. 2: 35. https://doi.org/10.3390/vetsci9020035