Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test.
Mycoplasma meleagridis, a turkey pathogen, can be detected by PCR directly from tracheal or genital swabs. However, up to 40% samples may contain inhibitory substances. A DNA fragment, that can be amplified with M. meleagridis primers and in the same cycling conditions, was constructed to use as an internal control (IC) to check for these inhibitors. This IC can easily be distinguished from the M. meleagridis amplicon after agarose gel electrophoresis since it is longer. Use of this IC in PCR amplifications revealed that more than 35% of turkey tracheal swabs and more than 45% of turkey cloacal swabs contained inhibitors. In most cases, dilution (1:100) of swab lysates allowed amplification of the internal control but DNA purification may be necessary to eliminate inhibitors (20% of tracheal swabs and 5% of cloacal swabs). Use of this internal control DNA allowed assessment of the efficiency of each individual reaction and ensured that the reaction was not inhibited by interfering substances.
Moalic
P Y
PY
U.R. Mycoplasmologie-Bactériologie, CNEVA-PLOUFRAGAN, ZOOPOLE, Ploufragan, France. Labofarm@wanadoo.fr
Gesbert
F
F
Kempf
I
I
D000818:Animals; D002988:Cloaca / Q000382:microbiology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D009174:Mycoplasma / Q000145:classification* / Q000302:isolation & purification*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D011201:Poultry Diseases; D015203:Reproducibility of Results; D014132:Trachea / Q000382:microbiology; D014422:Turkeys
D017931:DNA Primers; D004269:DNA, Bacterial