Development and comparison of loop-mediated isothermal amplification and quantitative polymerase chain reaction assays for the detection of Mycoplasma bovis in milk

摘要

Bovine mastitis is an economic burden for dairiesworldwide. Mycoplasma species, and especially Mycoplasmabovis, are among the most important causativeagents, and rapid, precise, and low-cost methods forMycoplasma detection are urgently needed. For this purpose,loop-mediated isothermal amplification (LAMP)and quantitative PCR (qPCR) assays were developedand compared. The LAMP assay was designed andprimer concentrations optimized to M. bovis oppD, encodingoligopeptide permease D. For qPCR, a Taqmanassay (Applied Biosystems, Carlsbad, CA) targeting M.bovis gltX, encoding glutamate transfer RNA ligase, wasoptimized for primer concentration, annealing temperature,and DNA polymerase. Both assays were similarlysensitive, with a detection limit of approximately 104 to105 M. bovis cells/mL. Both assays were also successfulin confirming M. bovis identity in laboratory culturesuspensions and in bovine milk. The LAMP and qPCRassays combined with the MoBio DNA extraction kit(MoBio Laboratories Inc., Carlsbad, CA) resulted inthe correct detection of 13 out of 13 M. bovis isolatesand 14 out of 16 M. bovis-positive milk samples collectedfrom commercial dairies in California. Whencombined with the PrepMan Ultra reagent (AppliedBiosystems), the qPCR assay resulted in confirming 21out of 21 M. bovis-positive milk samples. Comparison ofthe assays to milk containing either Mycoplasma arginini,Mycoplasma bovigenitalium, Mycoplasma californicum,M. alkalescens, or Acholeplasma laidlawii or milklacking any detectable Mycoplasma species or relativesresulted in 3 out of 17 (LAMP with MoBio), 1 out of17 (qPCR with MoBio), and 2 out of 36 (qPCR withPrepMan Ultra) false positives. Overall, the qPCR assaywas more robust than LAMP and could be used onDNA recovered from milk prepared with the PrepManUltra reagent, a method that does not include a DNApurification step. The use of this qPCR method enablesM. bovis detection in bovine milk in 40 to 55 min, andtherefore provides new opportunities to accelerate andsimplify M. bovis detection in unpasteurized milk toreduce the incidence of M. bovis mastitis outbreaks.