Abstract
Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 10 3 CFU/ml to 10 3 and 10 6 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 10 2 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
| Original language | English |
|---|---|
| Article number | 86 |
| Journal | BMC Veterinary Research |
| Volume | 15 |
| Issue number | 1 |
| Number of pages | 12 |
| ISSN | 1746-6148 |
| DOIs | |
| Publication status | Published - 2019 |
Keywords
- Bovine respiratory disease
- Bronchoalveolar lavage fluid
- Culture
- End-point PCR
- Mycoplasma bovis
- Real-time PCR
- Ring trial
Cite this
}
A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis. / Wisselink, Henk J.; Smid, Bregtje; Plater, Jane; Ridley, Anne; Andersson, Anna Maria; Aspán, Anna; Pohjanvirta, Tarja; Vähänikkilä, Nella; Larsen, Helene; Høgberg, Jonas; Colin, Adélie; Tardy, Florence.
In: BMC Veterinary Research, Vol. 15, No. 1, 86, 2019.Research output: Contribution to journal › Journal article › Research › peer-review
TY - JOUR
T1 - A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis
AU - Wisselink, Henk J.
AU - Smid, Bregtje
AU - Plater, Jane
AU - Ridley, Anne
AU - Andersson, Anna Maria
AU - Aspán, Anna
AU - Pohjanvirta, Tarja
AU - Vähänikkilä, Nella
AU - Larsen, Helene
AU - Høgberg, Jonas
AU - Colin, Adélie
AU - Tardy, Florence
PY - 2019
Y1 - 2019
N2 - Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 10 3 CFU/ml to 10 3 and 10 6 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 10 2 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
AB - Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 10 3 CFU/ml to 10 3 and 10 6 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 10 2 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
KW - Bovine respiratory disease
KW - Bronchoalveolar lavage fluid
KW - Culture
KW - End-point PCR
KW - Mycoplasma bovis
KW - Real-time PCR
KW - Ring trial
U2 - 10.1186/s12917-019-1819-7
DO - 10.1186/s12917-019-1819-7
M3 - Journal article
C2 - 30866933
AN - SCOPUS:85062829235
VL - 15
JO - B M C Veterinary Research
JF - B M C Veterinary Research
SN - 1746-6148
IS - 1
M1 - 86
ER -