Rapid, simple and cost-effective molecular method to differentiate the temperature sensitive (ts+) MS-H vaccine strain and wild-type Mycoplasma synoviae isolates

摘要

Mycoplasma synoviaeudinfection in chickens and turkeys can cause respiratory disease,udinfectious synovitis and eggshell apex abnormality; thus it is an economically importantudpathogen. Control ofudMud.udsynoviaeudinfection comprises eradication, medication or vaccina-udtion. The differentiation of the temperature sensitive (tsud+ud) MS-H vaccine strain from field iso-udlates is crucial during vaccination programs. Melt-curve and agarose gel based mismatchudamplification mutation assays (MAMA) are provided in the present study to distinguishudbetween the tsud+udMS-H vaccine strain, its non-temperature sensitive re-isolates and wild-udtypeudMud.udsynoviaeudisolates based on the single nucleotide polymorphisms at nt367 and nt629udof theudobgudgene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish theudtsud+udMS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotypeudand wild-typeudMud.udsynoviaeudisolate genotype, and no cross-reactions with otherudMycoplasmaudspecies infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs wasud10ud3udand 10ud4udcopy numbers, respectively. The assays can be performed directly on clinicaludsamples and they can be run simultaneously at the same annealing temperature. Theudassays can be performed in laboratories with limited facilities, using basic real-time PCRudmachine or conventional thermocycler coupled with agarose gel electrophoresis. Theudadvantages of the described assays compared with previously used methods are simplicity,udsufficient sensitivity, time and cost effectiveness and specificity.