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Utility of an internal control for evaluation of a Mycoplasma meleagridis PCR test
P Y Moalic1, F Gesbert, I Kempf
1U.R. Mycoplasmologie-Bactériologie, CNEVA-PLOUFRAGAN, ZOOPOLE, Ploufragan, France. Labofarm@wanadoo.fr
Abstract:
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.
Insights
This study introduces an internal control for Mycoplasma meleagridis PCR detection in turkeys. The control identifies inhibitors in swab samples, improving diagnostic accuracy for this turkey pathogen.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Avian Pathology
Background:
- Mycoplasma meleagridis is a significant turkey pathogen.
- PCR is a common method for detecting M. meleagridis in avian samples.
- PCR assays can be inhibited by substances present in biological samples, leading to false negatives.
Purpose of the Study:
- To develop and validate an internal control (IC) for PCR detection of Mycoplasma meleagridis.
- To assess the prevalence of PCR inhibitors in turkey tracheal and cloacal swabs.
- To improve the reliability of M. meleagridis PCR diagnostics in poultry.
Main Methods:
- Construction of a DNA fragment for use as an internal control (IC) amplified by M. meleagridis-specific primers.
- Incorporation of the IC into PCR reactions with turkey swab samples.
- Analysis of PCR products using agarose gel electrophoresis to differentiate IC and M. meleagridis amplicons.
- Evaluation of sample dilution and DNA purification strategies to overcome inhibition.
Main Results:
- The developed IC was successfully amplified under the same conditions as M. meleagridis.
- Over 35% of turkey tracheal swabs and 45% of cloacal swabs contained PCR inhibitors.
- Sample dilution (1:100) resolved inhibition in most cases.
- DNA purification was required for 20% of tracheal and 5% of cloacal swabs to enable amplification.
Conclusions:
- The internal control effectively detects PCR inhibitors in turkey swab samples.
- A significant proportion of turkey swabs contain substances that inhibit PCR.
- The use of this IC enhances the accuracy and reliability of Mycoplasma meleagridis detection in veterinary diagnostics.