Interlaboratory comparison of ability to detect nucleic acid of Mycoplasma gallisepticum and Mycoplasma synoviae by polymerase chain reaction

Abstract

In recent years polymerase chain reaction (PCR) assays have become widely used as methods to confirm the presence of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry flocks, but there has been limited standardization of the protocols used. Thirteen laboratories from five different countries participated in an interlaboratory comparison of detection of M. gallisepticum and M. synoviae DNA by PCR in samples that contained 10-fold dilutions of these bacteria. The concentration of bacteria ranged from 105 to 102 genome copies/100 µl sample, as quantified by real-time PCR, and the samples were supplied on dry cotton swabs. Each laboratory was asked to use its standard method for PCR testing of these pathogens. A questionnaire was supplied with the samples to obtain details of the methods that were used in testing. One-half of the laboratories used a commercially available test kit, while the others used an in-house protocol. The protocols used for DNA extraction varied greatly, even among those using commercially available test kits. Two laboratories had developed the primers for nucleic acid amplification themselves, and one of these used real-time PCR for amplification. While the majority of the laboratories detected M. synoviae down to the 100 copy limit of the comparison, the detection limit for M. gallisepticum was somewhat higher. Furthermore, different results were obtained from laboratories that used the same commercial test kit. To the best of our knowledge this is the first investigation of its kind in the field of avian diseases.