A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

Henk J. Wisselink*, Bregtje Smid, Jane Plater, Anne Ridley, Anna Maria Andersson, Anna Aspán, Tarja Pohjanvirta, Nella Vähänikkilä, Helene Larsen, Jonas Høgberg, Adélie Colin, Florence Tardy

*Corresponding author for this work

    Research output: Contribution to journalJournal articleResearchpeer-review

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    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 languageEnglish
    Article number86
    JournalBMC Veterinary Research
    Volume15
    Issue number1
    Number of pages12
    ISSN1746-6148
    DOIs
    Publication statusPublished - 2019

    Keywords

    • Bovine respiratory disease
    • Bronchoalveolar lavage fluid
    • Culture
    • End-point PCR
    • Mycoplasma bovis
    • Real-time PCR
    • Ring trial

    Cite this

    Wisselink, H. J., Smid, B., Plater, J., Ridley, A., Andersson, A. M., Aspán, A., ... Tardy, F. (2019). A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis. BMC Veterinary Research, 15(1), [86]. https://doi.org/10.1186/s12917-019-1819-7
    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. / A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis. In: BMC Veterinary Research. 2019 ; Vol. 15, No. 1.
    @article{a06036cab875481d911fda822008568b,
    title = "A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis",
    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.",
    keywords = "Bovine respiratory disease, Bronchoalveolar lavage fluid, Culture, End-point PCR, Mycoplasma bovis, Real-time PCR, Ring trial",
    author = "Wisselink, {Henk J.} and Bregtje Smid and Jane Plater and Anne Ridley and Andersson, {Anna Maria} and Anna Asp{\'a}n and Tarja Pohjanvirta and Nella V{\"a}h{\"a}nikkil{\"a} and Helene Larsen and Jonas H{\o}gberg and Ad{\'e}lie Colin and Florence Tardy",
    year = "2019",
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    journal = "B M C Veterinary Research",
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    Wisselink, HJ, Smid, B, Plater, J, Ridley, A, Andersson, AM, Aspán, A, Pohjanvirta, T, Vähänikkilä, N, Larsen, H, Høgberg, J, Colin, A & Tardy, F 2019, 'A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis', BMC Veterinary Research, vol. 15, no. 1, 86. https://doi.org/10.1186/s12917-019-1819-7

    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 journalJournal articleResearchpeer-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 -