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

H.J. Wisselink, B. Smid, J. Plater, A. Ridley, Anna-Maria Andersson, A. Aspán, T. Pohjanvirta, Nella Vähänikkilä, Helena Larsen, Jonas Hogberg, A. Colin, Florence Tardy

Research output: Contribution to journalArticleAcademicpeer-review

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 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 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.
LanguageEnglish
Article number86
JournalBMC Veterinary Research
Volume15
DOIs
Publication statusPublished - 12 Mar 2019

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Mycoplasma bovis
Polymerase Chain Reaction
analytical specificity
methodology
Mycoplasma agalactiae
detection limit
assays
Endpoint Determination
Mycoplasma Infections
sampling
veal calves
Workflow
Mastitis
Mycoplasma
DNA
respiratory tract diseases
Genes
mastitis
Limit of Detection
Real-Time Polymerase Chain Reaction

Cite this

Wisselink, H.J. ; Smid, B. ; Plater, J. ; Ridley, A. ; Andersson, Anna-Maria ; Aspán, A. ; Pohjanvirta, T. ; Vähänikkilä, Nella ; Larsen, Helena ; Hogberg, Jonas ; Colin, A. ; 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.
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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 diversityof 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 nationalveterinary 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 onecommercial 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 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 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.",
author = "H.J. Wisselink and B. Smid and J. Plater and A. Ridley and Anna-Maria Andersson and A. Asp{\'a}n and T. Pohjanvirta and Nella V{\"a}h{\"a}nikkil{\"a} and Helena Larsen and Jonas Hogberg and A. Colin and Florence Tardy",
year = "2019",
month = "3",
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Wisselink, HJ, Smid, B, Plater, J, Ridley, A, Andersson, A-M, Aspán, A, Pohjanvirta, T, Vähänikkilä, N, Larsen, H, Hogberg, 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, 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, H.J.; Smid, B.; Plater, J.; Ridley, A.; Andersson, Anna-Maria; Aspán, A.; Pohjanvirta, T.; Vähänikkilä, Nella; Larsen, Helena; Hogberg, Jonas; Colin, A.; Tardy, Florence .

In: BMC Veterinary Research, Vol. 15, 86, 12.03.2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

AU - Wisselink, H.J.

AU - Smid, B.

AU - Plater, J.

AU - Ridley, A.

AU - Andersson, Anna-Maria

AU - Aspán, A.

AU - Pohjanvirta, T.

AU - Vähänikkilä, Nella

AU - Larsen, Helena

AU - Hogberg, Jonas

AU - Colin, A.

AU - Tardy, Florence

PY - 2019/3/12

Y1 - 2019/3/12

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 diversityof 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 nationalveterinary 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 onecommercial 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 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 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 diversityof 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 nationalveterinary 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 onecommercial 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 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 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.

U2 - 10.1186/s12917-019-1819-7

DO - 10.1186/s12917-019-1819-7

M3 - Article

VL - 15

JO - BMC Veterinary Research

T2 - BMC Veterinary Research

JF - BMC Veterinary Research

SN - 1746-6148

M1 - 86

ER -