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 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.

Keywords: Bovine respiratory disease; Bronchoalveolar lavage fluid; Culture; End-point PCR; Mycoplasma bovis; Real-time PCR; Ring trial.

Fig. 1

Determination of the analytical specificity of various M. bovis PCR assays in use by six laboratories (Lab 1 to Lab 6) on DNA from a panel of seven M. bovis strains and 10 non-target mycoplasma species. DNA of each strain was tested in two concentrations: once with 5 μl of 10 ng/μl (pure) and twice with 5 μl of a 1/10 dilution (1:10), except for Lab 4 that used 1 μl of DNA only. Raw data to this figure are provided in Additional file 1

Fig. 2

Determination of the analytical sensitivity of various M. bovis PCR assays in use by six laboratories (Lab 1 to Lab 6) on spiked bronchoalveolar lavage fluid (BALF) samples. Ten-fold serial dilutions (n = 7) of a mixture of 1 × 10⁸ M. bovis CFU/ml were prepared in BALF resulting in a range of 1 × 10⁷ to 1 × 10¹ M. bovis CFU/ml and tested by the various PCR methods. Viability of M. bovis in the BALF samples was verified with culture. Raw data to this figure are provided in Additional file 1

Fig. 3

Comparability of results of various PCR methods in use by six laboratories (Lab 1 to Lab 6) on bronchoalveolar lavage fluid (BALF) samples (n = 21) from veal calves from farms with bovine respiratory disease (BRD). BALF samples were tested twice by the PCR methods and the mean Ct values were calculated from the real-time PCRs. Viability of M. bovis in the BALF samples was verified with culture. On the basis of the culture results, the figure was split in two parts, either with BALF samples negative or positive for M. bovis by culture. Raw data to this figure are provided in Additional file 1