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Korean J. Vet. Serv. 2023; 46(1): 15-27
Published online March 30, 2023
https://doi.org/10.7853/kjvs.2023.46.1.15
© The Korean Socitety of Veterinary Service
Correspondence to : Choi-Kyu Park
E-mail: parkck@knu.ac.kr
https://orcid.org/0000-0002-0784-9061
†These first two authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0). which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Bordetella (B.) bronchiseptica, Mycoplasma (M.) cynos, and M. canis are the major bacterial pathogens that cause canine infectious respiratory disease complex (CIRDC). In this study, we developed a triplex real-time polymerase chain reaction (tqPCR) assay for the differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra- and inter-assay variations of less than 1%. The diagnostic results of the assay using 94 clinical samples from household dogs with CIRDC clinical signs, the prevalence of B. bronchiseptica, M. cynos, and M. canis was 22.3%, 18.1%, and 20.2%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported qPCR assays. The dual infection rate of B. bronchiseptica and M. cynos, B. bronchiseptica and M. canis, and M. cynos and M. canis was 5.3%, 7.4%, and 3.1%, respectively. Moreover, the triple infection rate of B. bronchiseptica, M. cynos, and M. canis was 2.1%. These results indicate that coinfections with B. bronchiseptica, M. cynos, and M. canis have frequently occurred in the Korean dog population. The newly developed tqPCR assay in the present study will be a useful tool for etiological and epidemiological studies on these three CIRDC-associated bacterial pathogens. The prevalence and coinfection data revealed through this study will contribute to expanding knowledge on the epidemiology of CIRDC in the recent Korean dog population.
Keywords Triplex real-time PCR, Dogs, Bordetella bronchiseptica, Mycoplasma cynos, Mycoplasma canis
Canine infectious respiratory disease complex (CIRDC) is a multifactorial respiratory disease syndrome in the global dog population and is caused by various etiological agents. The primary pathogens associated with CIRDC include viral pathogens, such as canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), canine parainfluenza virus 5 (CPIV5), and canine respiratory coronavirus (CRCoV) as well as bacterial pathogens, including
To date, more than 15 different
Since these three bacteria are frequently associated with CIRDC, several conventional polymerase chain reaction (cPCR) and quantitative real-time PCR (qPCR) assays have been developed for their rapid, sensitive, and specific detection in suspected clinical samples. TaqMan probe-based qPCR assays are currently preferred because of their superior sensitivity, specificity, and reliability over cPCR assays (Chalker et al, 2004; Helps et al, 2005; Windsor et al, 2006; Spergser and Rosengarten, 2007; Tizolova et al, 2014; Jinnerot et al, 2015; Maboni et al, 2019; Matsuu et al, 2020; Tallmadge et al, 2020; Dong et al, 2022). So far, several TaqMan probe-based qPCR assays have been developed in a monoplex format for the individual detection of
Table 1 . Specificity of the triplex real-time polymerase chain reaction using different canine pathogens and controls
Pathogen | Strain | Source* | Amplification of target gene† | ||
---|---|---|---|---|---|
Bb (FAM) | Mcy (Texas red) | Mca (Cy5) | |||
92b | CAVS | + | − | − | |
Field strain | ADIC | − | + | − | |
PG14 | ADIC | − | − | + | |
Canine distemper virus | Onderstepoort | CAVS | − | − | − |
Canine influenza virus | A/Canine/Korea/01/07(H3N2) | CAVS | − | − | − |
Canine coronavirus | NL-18 | CAVS | − | − | − |
Canine parainfluenza virus 5 | D008 | CAVS | − | − | − |
Canine parvovirus | 7809 16-LP | CAVS | − | − | − |
Canine adenovirus 2 | Ditchfield | CAVS | − | − | − |
Canine pneumovirus | Dog/Bari/100-12/ITA/2012 | ADIC | − | − | − |
MDCK cell | - | ADIC | − | − | − |
*CAVS, commercially available vaccine strain; ADIC, Animal Disease Intervention Center, Kyungpook National University, Korea.
†Each probe labeled with FAM, Texas red, and Cy5 fluorescent dye detected flaA gene of
In the tqPCR assay, three sets of primers and probes were used for the differential detection of
Table 2 . Primers and probes for the triplex real-time polymerase chain reaction
Method | Pathogen/gene | Primer/probe | Sequence (5’–3’)† | Tm (℃) | Amplicon (bp) | Reference |
---|---|---|---|---|---|---|
tqPCR | Bb/ | Bb-F | GAACTCGGCTTCGGACATC | 62.1 | 116 | Kim et al. (2022) |
Bb-R | CGTTGGACTTCAGGACCTTG | 62.3 | ||||
Bb-P | FAM-TCTGCTCGGCGATGCGGTTGATTTC-BHQ1 | 70.4 | ||||
Mcy/ | Fla-F | CCTCCTTTCTACGGAGTACA | 60.1 | 144 | This study | |
Mcy-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mcy-P | Texas red-AATGTTGTGTTTATGTATCTAGTTTTGAGAGAAC-BHQ2 | 65.0 | ||||
Mca/ | Mca-F | CCTCCTTTCTACGGAGTACA | 60.1 | 166 | This study | |
Mca-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mca-P | Cy5-AATGTCATGTGTCGAGATAACCCGACAGT-BHQ3 | 68.8 | ||||
qPCR | Bb/ | Fla2 | AGGCTCCCAAGAGAGAAGGCTT | 67.0 | 118 | Tizolova et al. (2014) |
Fla12 | AAACCTGCCGTAATCCAGGC | 64.7 | ||||
Fla-FAM3 | FAM-ACCGGGCAGCTAGGCCGC-BHQ1 | 71.1 | ||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-AAGCAAAATGGTGACATCAAGCA-BHQ1 | 63.9 | |||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-CAAAACGGCGACGTCAAGC-BHQ1 | 65.1 |
†Primers and probes for the assays were designed based on the sequences of
Plasmids containing the target genes of
Prior to the optimization of the tqPCR conditions, monoplex qPCR using a
To evaluate the specificity of the assay, tqPCR was performed using total nucleic acid samples extracted from 10 canine pathogens (
The repeatability (intra-assay precision) and reproducibility (inter-assay precision) of the tqPCR assay for detecting
Reference qPCR assays for
To evaluate the diagnostic performance of the tqPCR assay for the differential detection of
For the simultaneous and differential detection of
Each set of primers and probes for
The coefficient of variation within runs (intra-assay variability) was 0.12%∼0.44% for
Table 3 . Intra- and inter-assay coefficients of variation of the triplex real-time polymerase chain reaction for
Pathogen | Dilution (copies/reaction) | Intra-assay variability | Inter-assay variability | |||||
---|---|---|---|---|---|---|---|---|
Mean | SD | CV (%) | Mean | SD | CV (%) | |||
High (106) | 19.32 | 0.02 | 0.12 | 20.09 | 0.07 | 0.35 | ||
Medium (104) | 26.16 | 0.11 | 0.44 | 26.80 | 0.15 | 0.55 | ||
Low (102) | 33.14 | 0.14 | 0.43 | 33.68 | 0.22 | 0.66 | ||
High (106) | 19.14 | 0.07 | 0.36 | 18.23 | 0.06 | 0.33 | ||
Medium (104) | 26.06 | 0.11 | 0.41 | 25.08 | 0.09 | 0.34 | ||
Low (102) | 32.33 | 0.27 | 0.82 | 31.77 | 0.09 | 0.27 | ||
High (106) | 18.35 | 0.06 | 0.30 | 18.04 | 0.11 | 0.59 | ||
Medium (104) | 25.06 | 0.14 | 0.54 | 25.39 | 0.17 | 0.69 | ||
Low (102) | 32.04 | 0.09 | 0.30 | 32.03 | 0.27 | 0.83 |
The mean value, standard deviation (SD), and coefficient of variation (CV) were determined based on the Ct values for the tqPCR assay.
For the clinical evaluation of the newly developed tqPCR assay and investigation of the prevalence and coinfection status of
Table 4 . Comparison of diagnostic results between the novel triplex real-time polymerase chain reaction (tqPCR) and previous qPCR assays for detecting
Methods | Pathogen | No. of tested | No. of positive | Detection rate (%)† |
---|---|---|---|---|
New tqPCR | 94 | 21 | 22.3 | |
94 | 17 | 18.1 | ||
94 | 19 | 20.2 | ||
Previous qPCR | 94 | 21 | 22.3 | |
94 | 16 | 17.0 | ||
94 | 19 | 20.2 |
*One clinical sample that tested negative for
†The positive, negative, and overall agreements between the newly developed tqPCR and previously described qPCR assays were all 100% for
CIRDC, also known as kennel cough, is an endemic syndrome caused by multiple viral and bacterial pathogens, including CDV, CPIV5, CAdV-2, CHV-1, CPIV5, CRCoV,
The newly developed tqPCR assay has some advantages. First, it can specifically and differentially detect the three main bacterial pathogens associated with CIRDC in a single reaction without any cross-reactivity with other canine pathogens (Table 1 and Fig. 1). Second, the sensitivity of the newly developed tqPCR assay was <10 copies/reaction with the standard DNAs of
Despite the endemic occurrence of
Notably, rates of dual or triple infections with
This study has some limitations. First, since this study aimed to development of the tqPCR assay for the three main bacteria associated with CIRDC and to investigate their prevalence, major viral pathogens associated with CIRDC were not included in the scope of the study. Second, the clinical samples used in this study were collected from household dogs who visited animal clinics. But no samples were obtained from dogs in animal shelters, an important subpopulation in which the prevalence of CIRDC should be investigated. Therefore, further studies are required to develop more advanced diagnostic assays for various canine pathogens associated with CIRDC and investigate its prevalence in a larger representative sample of the entire Korean dog population including animal shelters.
Conclusively, we developed a tqPCR assay that can simultaneously detect three main bacterial pathogens associated with CIRDC in a single reaction. Based on the clinical diagnostic results of the assay, the prevalence and co-infection status of
This work was supported by the fund (Z-1543085-2022- 23-03) by the Research of Animal and Plant Quarantine Agency, Republic of Korea.
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. This study was conducted in 2022 and was beyond the purview of the Institutional Animal Care and Use Committee (IACUC) at Kyungpook National University (KNU), as the KNU IACUC only evaluates proposals using laboratory animals maintained in indoor facilities and not research involving outdoor animals. Canine and feline clinical samples were collected by practicing veterinarians at local clinics and animal shelters during monitoring, surveillance, and treatment, or during regular medical check-ups, after receiving verbal consent from the owners.
No potential conflict of interest relevant to this article was reported.
Korean J. Vet. Serv. 2023; 46(1): 15-27
Published online March 30, 2023 https://doi.org/10.7853/kjvs.2023.46.1.15
Copyright © The Korean Socitety of Veterinary Service.
Gyu-Tae Jeon 1†, Jong-Min Kim
1†, Jeong-Hyun Park
1, Hye-Ryung Kim
1, Ji-Su Baek
1, Hyo-Ji Lee
1, Yeun-Kyung Shin
2, Oh-Kyu Kwon
2, Hae-Eun Kang
2, Soong-Koo Kim
3, Jung-Hwa Kim
3, Young-Hwan Kim
3, Choi-Kyu Park
1*
1College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University, Daegu 41566, Korea
2Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
3Gyeongsangbuk-do Veterinary Service Laboratory, Daegu 41405, Korea
Correspondence to:Choi-Kyu Park
E-mail: parkck@knu.ac.kr
https://orcid.org/0000-0002-0784-9061
†These first two authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0). which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Bordetella (B.) bronchiseptica, Mycoplasma (M.) cynos, and M. canis are the major bacterial pathogens that cause canine infectious respiratory disease complex (CIRDC). In this study, we developed a triplex real-time polymerase chain reaction (tqPCR) assay for the differential detection of these bacteria in a single reaction. The assay specifically amplified three bacterial genes with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra- and inter-assay variations of less than 1%. The diagnostic results of the assay using 94 clinical samples from household dogs with CIRDC clinical signs, the prevalence of B. bronchiseptica, M. cynos, and M. canis was 22.3%, 18.1%, and 20.2%, respectively, indicating that the diagnostic sensitivity was comparable to those of previously reported qPCR assays. The dual infection rate of B. bronchiseptica and M. cynos, B. bronchiseptica and M. canis, and M. cynos and M. canis was 5.3%, 7.4%, and 3.1%, respectively. Moreover, the triple infection rate of B. bronchiseptica, M. cynos, and M. canis was 2.1%. These results indicate that coinfections with B. bronchiseptica, M. cynos, and M. canis have frequently occurred in the Korean dog population. The newly developed tqPCR assay in the present study will be a useful tool for etiological and epidemiological studies on these three CIRDC-associated bacterial pathogens. The prevalence and coinfection data revealed through this study will contribute to expanding knowledge on the epidemiology of CIRDC in the recent Korean dog population.
Keywords: Triplex real-time PCR, Dogs, Bordetella bronchiseptica, Mycoplasma cynos, Mycoplasma canis
Canine infectious respiratory disease complex (CIRDC) is a multifactorial respiratory disease syndrome in the global dog population and is caused by various etiological agents. The primary pathogens associated with CIRDC include viral pathogens, such as canine distemper virus (CDV), canine adenovirus 2 (CAdV-2), canine parainfluenza virus 5 (CPIV5), and canine respiratory coronavirus (CRCoV) as well as bacterial pathogens, including
To date, more than 15 different
Since these three bacteria are frequently associated with CIRDC, several conventional polymerase chain reaction (cPCR) and quantitative real-time PCR (qPCR) assays have been developed for their rapid, sensitive, and specific detection in suspected clinical samples. TaqMan probe-based qPCR assays are currently preferred because of their superior sensitivity, specificity, and reliability over cPCR assays (Chalker et al, 2004; Helps et al, 2005; Windsor et al, 2006; Spergser and Rosengarten, 2007; Tizolova et al, 2014; Jinnerot et al, 2015; Maboni et al, 2019; Matsuu et al, 2020; Tallmadge et al, 2020; Dong et al, 2022). So far, several TaqMan probe-based qPCR assays have been developed in a monoplex format for the individual detection of
Table 1 . Specificity of the triplex real-time polymerase chain reaction using different canine pathogens and controls.
Pathogen | Strain | Source* | Amplification of target gene† | ||
---|---|---|---|---|---|
Bb (FAM) | Mcy (Texas red) | Mca (Cy5) | |||
92b | CAVS | + | − | − | |
Field strain | ADIC | − | + | − | |
PG14 | ADIC | − | − | + | |
Canine distemper virus | Onderstepoort | CAVS | − | − | − |
Canine influenza virus | A/Canine/Korea/01/07(H3N2) | CAVS | − | − | − |
Canine coronavirus | NL-18 | CAVS | − | − | − |
Canine parainfluenza virus 5 | D008 | CAVS | − | − | − |
Canine parvovirus | 7809 16-LP | CAVS | − | − | − |
Canine adenovirus 2 | Ditchfield | CAVS | − | − | − |
Canine pneumovirus | Dog/Bari/100-12/ITA/2012 | ADIC | − | − | − |
MDCK cell | - | ADIC | − | − | − |
*CAVS, commercially available vaccine strain; ADIC, Animal Disease Intervention Center, Kyungpook National University, Korea..
†Each probe labeled with FAM, Texas red, and Cy5 fluorescent dye detected flaA gene of
In the tqPCR assay, three sets of primers and probes were used for the differential detection of
Table 2 . Primers and probes for the triplex real-time polymerase chain reaction.
Method | Pathogen/gene | Primer/probe | Sequence (5’–3’)† | Tm (℃) | Amplicon (bp) | Reference |
---|---|---|---|---|---|---|
tqPCR | Bb/ | Bb-F | GAACTCGGCTTCGGACATC | 62.1 | 116 | Kim et al. (2022) |
Bb-R | CGTTGGACTTCAGGACCTTG | 62.3 | ||||
Bb-P | FAM-TCTGCTCGGCGATGCGGTTGATTTC-BHQ1 | 70.4 | ||||
Mcy/ | Fla-F | CCTCCTTTCTACGGAGTACA | 60.1 | 144 | This study | |
Mcy-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mcy-P | Texas red-AATGTTGTGTTTATGTATCTAGTTTTGAGAGAAC-BHQ2 | 65.0 | ||||
Mca/ | Mca-F | CCTCCTTTCTACGGAGTACA | 60.1 | 166 | This study | |
Mca-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mca-P | Cy5-AATGTCATGTGTCGAGATAACCCGACAGT-BHQ3 | 68.8 | ||||
qPCR | Bb/ | Fla2 | AGGCTCCCAAGAGAGAAGGCTT | 67.0 | 118 | Tizolova et al. (2014) |
Fla12 | AAACCTGCCGTAATCCAGGC | 64.7 | ||||
Fla-FAM3 | FAM-ACCGGGCAGCTAGGCCGC-BHQ1 | 71.1 | ||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-AAGCAAAATGGTGACATCAAGCA-BHQ1 | 63.9 | |||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-CAAAACGGCGACGTCAAGC-BHQ1 | 65.1 |
†Primers and probes for the assays were designed based on the sequences of
Plasmids containing the target genes of
Prior to the optimization of the tqPCR conditions, monoplex qPCR using a
To evaluate the specificity of the assay, tqPCR was performed using total nucleic acid samples extracted from 10 canine pathogens (
The repeatability (intra-assay precision) and reproducibility (inter-assay precision) of the tqPCR assay for detecting
Reference qPCR assays for
To evaluate the diagnostic performance of the tqPCR assay for the differential detection of
For the simultaneous and differential detection of
Each set of primers and probes for
The coefficient of variation within runs (intra-assay variability) was 0.12%∼0.44% for
Table 3 . Intra- and inter-assay coefficients of variation of the triplex real-time polymerase chain reaction for
Pathogen | Dilution (copies/reaction) | Intra-assay variability | Inter-assay variability | |||||
---|---|---|---|---|---|---|---|---|
Mean | SD | CV (%) | Mean | SD | CV (%) | |||
High (106) | 19.32 | 0.02 | 0.12 | 20.09 | 0.07 | 0.35 | ||
Medium (104) | 26.16 | 0.11 | 0.44 | 26.80 | 0.15 | 0.55 | ||
Low (102) | 33.14 | 0.14 | 0.43 | 33.68 | 0.22 | 0.66 | ||
High (106) | 19.14 | 0.07 | 0.36 | 18.23 | 0.06 | 0.33 | ||
Medium (104) | 26.06 | 0.11 | 0.41 | 25.08 | 0.09 | 0.34 | ||
Low (102) | 32.33 | 0.27 | 0.82 | 31.77 | 0.09 | 0.27 | ||
High (106) | 18.35 | 0.06 | 0.30 | 18.04 | 0.11 | 0.59 | ||
Medium (104) | 25.06 | 0.14 | 0.54 | 25.39 | 0.17 | 0.69 | ||
Low (102) | 32.04 | 0.09 | 0.30 | 32.03 | 0.27 | 0.83 |
The mean value, standard deviation (SD), and coefficient of variation (CV) were determined based on the Ct values for the tqPCR assay..
For the clinical evaluation of the newly developed tqPCR assay and investigation of the prevalence and coinfection status of
Table 4 . Comparison of diagnostic results between the novel triplex real-time polymerase chain reaction (tqPCR) and previous qPCR assays for detecting
Methods | Pathogen | No. of tested | No. of positive | Detection rate (%)† |
---|---|---|---|---|
New tqPCR | 94 | 21 | 22.3 | |
94 | 17 | 18.1 | ||
94 | 19 | 20.2 | ||
Previous qPCR | 94 | 21 | 22.3 | |
94 | 16 | 17.0 | ||
94 | 19 | 20.2 |
*One clinical sample that tested negative for
†The positive, negative, and overall agreements between the newly developed tqPCR and previously described qPCR assays were all 100% for
CIRDC, also known as kennel cough, is an endemic syndrome caused by multiple viral and bacterial pathogens, including CDV, CPIV5, CAdV-2, CHV-1, CPIV5, CRCoV,
The newly developed tqPCR assay has some advantages. First, it can specifically and differentially detect the three main bacterial pathogens associated with CIRDC in a single reaction without any cross-reactivity with other canine pathogens (Table 1 and Fig. 1). Second, the sensitivity of the newly developed tqPCR assay was <10 copies/reaction with the standard DNAs of
Despite the endemic occurrence of
Notably, rates of dual or triple infections with
This study has some limitations. First, since this study aimed to development of the tqPCR assay for the three main bacteria associated with CIRDC and to investigate their prevalence, major viral pathogens associated with CIRDC were not included in the scope of the study. Second, the clinical samples used in this study were collected from household dogs who visited animal clinics. But no samples were obtained from dogs in animal shelters, an important subpopulation in which the prevalence of CIRDC should be investigated. Therefore, further studies are required to develop more advanced diagnostic assays for various canine pathogens associated with CIRDC and investigate its prevalence in a larger representative sample of the entire Korean dog population including animal shelters.
Conclusively, we developed a tqPCR assay that can simultaneously detect three main bacterial pathogens associated with CIRDC in a single reaction. Based on the clinical diagnostic results of the assay, the prevalence and co-infection status of
This work was supported by the fund (Z-1543085-2022- 23-03) by the Research of Animal and Plant Quarantine Agency, Republic of Korea.
The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. This study was conducted in 2022 and was beyond the purview of the Institutional Animal Care and Use Committee (IACUC) at Kyungpook National University (KNU), as the KNU IACUC only evaluates proposals using laboratory animals maintained in indoor facilities and not research involving outdoor animals. Canine and feline clinical samples were collected by practicing veterinarians at local clinics and animal shelters during monitoring, surveillance, and treatment, or during regular medical check-ups, after receiving verbal consent from the owners.
No potential conflict of interest relevant to this article was reported.
Table 1 . Specificity of the triplex real-time polymerase chain reaction using different canine pathogens and controls.
Pathogen | Strain | Source* | Amplification of target gene† | ||
---|---|---|---|---|---|
Bb (FAM) | Mcy (Texas red) | Mca (Cy5) | |||
92b | CAVS | + | − | − | |
Field strain | ADIC | − | + | − | |
PG14 | ADIC | − | − | + | |
Canine distemper virus | Onderstepoort | CAVS | − | − | − |
Canine influenza virus | A/Canine/Korea/01/07(H3N2) | CAVS | − | − | − |
Canine coronavirus | NL-18 | CAVS | − | − | − |
Canine parainfluenza virus 5 | D008 | CAVS | − | − | − |
Canine parvovirus | 7809 16-LP | CAVS | − | − | − |
Canine adenovirus 2 | Ditchfield | CAVS | − | − | − |
Canine pneumovirus | Dog/Bari/100-12/ITA/2012 | ADIC | − | − | − |
MDCK cell | - | ADIC | − | − | − |
*CAVS, commercially available vaccine strain; ADIC, Animal Disease Intervention Center, Kyungpook National University, Korea..
†Each probe labeled with FAM, Texas red, and Cy5 fluorescent dye detected flaA gene of
Table 2 . Primers and probes for the triplex real-time polymerase chain reaction.
Method | Pathogen/gene | Primer/probe | Sequence (5’–3’)† | Tm (℃) | Amplicon (bp) | Reference |
---|---|---|---|---|---|---|
tqPCR | Bb/ | Bb-F | GAACTCGGCTTCGGACATC | 62.1 | 116 | Kim et al. (2022) |
Bb-R | CGTTGGACTTCAGGACCTTG | 62.3 | ||||
Bb-P | FAM-TCTGCTCGGCGATGCGGTTGATTTC-BHQ1 | 70.4 | ||||
Mcy/ | Fla-F | CCTCCTTTCTACGGAGTACA | 60.1 | 144 | This study | |
Mcy-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mcy-P | Texas red-AATGTTGTGTTTATGTATCTAGTTTTGAGAGAAC-BHQ2 | 65.0 | ||||
Mca/ | Mca-F | CCTCCTTTCTACGGAGTACA | 60.1 | 166 | This study | |
Mca-R | CTTTACTATTCAGTTTTCAAAGAACA | 59.2 | ||||
Mca-P | Cy5-AATGTCATGTGTCGAGATAACCCGACAGT-BHQ3 | 68.8 | ||||
qPCR | Bb/ | Fla2 | AGGCTCCCAAGAGAGAAGGCTT | 67.0 | 118 | Tizolova et al. (2014) |
Fla12 | AAACCTGCCGTAATCCAGGC | 64.7 | ||||
Fla-FAM3 | FAM-ACCGGGCAGCTAGGCCGC-BHQ1 | 71.1 | ||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-AAGCAAAATGGTGACATCAAGCA-BHQ1 | 63.9 | |||||
CAACACACGTGCTACAATGGA | 60.6 | 96 | Dong et al. (2022) | |||
TGCAGACTACAATCCGAACTGA | 60.8 | |||||
FAM-CAAAACGGCGACGTCAAGC-BHQ1 | 65.1 |
†Primers and probes for the assays were designed based on the sequences of
Table 3 . Intra- and inter-assay coefficients of variation of the triplex real-time polymerase chain reaction for
Pathogen | Dilution (copies/reaction) | Intra-assay variability | Inter-assay variability | |||||
---|---|---|---|---|---|---|---|---|
Mean | SD | CV (%) | Mean | SD | CV (%) | |||
High (106) | 19.32 | 0.02 | 0.12 | 20.09 | 0.07 | 0.35 | ||
Medium (104) | 26.16 | 0.11 | 0.44 | 26.80 | 0.15 | 0.55 | ||
Low (102) | 33.14 | 0.14 | 0.43 | 33.68 | 0.22 | 0.66 | ||
High (106) | 19.14 | 0.07 | 0.36 | 18.23 | 0.06 | 0.33 | ||
Medium (104) | 26.06 | 0.11 | 0.41 | 25.08 | 0.09 | 0.34 | ||
Low (102) | 32.33 | 0.27 | 0.82 | 31.77 | 0.09 | 0.27 | ||
High (106) | 18.35 | 0.06 | 0.30 | 18.04 | 0.11 | 0.59 | ||
Medium (104) | 25.06 | 0.14 | 0.54 | 25.39 | 0.17 | 0.69 | ||
Low (102) | 32.04 | 0.09 | 0.30 | 32.03 | 0.27 | 0.83 |
The mean value, standard deviation (SD), and coefficient of variation (CV) were determined based on the Ct values for the tqPCR assay..
Table 4 . Comparison of diagnostic results between the novel triplex real-time polymerase chain reaction (tqPCR) and previous qPCR assays for detecting
Methods | Pathogen | No. of tested | No. of positive | Detection rate (%)† |
---|---|---|---|---|
New tqPCR | 94 | 21 | 22.3 | |
94 | 17 | 18.1 | ||
94 | 19 | 20.2 | ||
Previous qPCR | 94 | 21 | 22.3 | |
94 | 16 | 17.0 | ||
94 | 19 | 20.2 |
*One clinical sample that tested negative for
†The positive, negative, and overall agreements between the newly developed tqPCR and previously described qPCR assays were all 100% for
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