A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA.
OBJECTIVE
The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples.


RESULTS
A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2.


CONCLUSIONS
The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week).


CONCLUSIONS
The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.
Lorusso
A
A
Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, Valenzano (Bari), Italy.
Decaro
N
N
Greco
G
G
Corrente
M
M
Fasanella
A
A
Buonavoglia
D
D
D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis*; D008892:Milk / Q000382:microbiology; D009175:Mycoplasma Infections / Q000382:microbiology / Q000662:veterinary*; D045644:Mycoplasma agalactiae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*
D004269:DNA, Bacterial