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Updated: Jul 16, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Improved template DNA preparation procedure for detection of Mycobacterium avium subsp. paratuberculosis in milk by
Anli Gao1, Lucy Mutharia, Melinda Raymond
1Laboratory Services Division (Gao, Odumeru), University of Guelph, 95 Stone Rd W, Guelph, ON Canada N1H 8J7.
Abstract:
Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15-50 CFU in 50 ml, or
Insights
Optimizing Mycobacterium avium subsp. paratuberculosis (MAP) detection in raw milk using PCR involves specific pre-treatment steps. Heat treatment and hexadecylpyridinium chloride (HPC) significantly improve MAP detection limits in raw milk samples.
Area of Science:
- Food microbiology
- Veterinary diagnostics
- Molecular biology
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen in cattle.
- Accurate detection of MAP in raw milk is crucial for controlling its spread.
- Current PCR methods for MAP detection in raw milk can be affected by various factors.
Purpose of the Study:
- To investigate factors influencing the detection of MAP by PCR in raw milk.
- To optimize a procedure for enhanced MAP detection in raw milk samples.
- To determine the limit of detection for MAP in raw milk using an optimized PCR method.
Main Methods:
- Raw milk samples (50 ml) were artificially contaminated with MAP (approx. 30 CFU/ml).
- Investigated effects of heat treatment, hexadecylpyridinium chloride (HPC) treatment, and sample fractionation (cream, whey, pellet) on MAP partitioning.
- Assessed the impact of pooling fractions and washing pellets on PCR detection efficiency.
- Determined the limit of detection (LOD) using IS900 PCR.
Main Results:
- Heat treatment significantly altered MAP partitioning, with MAP found in the cream fraction of unheated milk and the pellet fraction of heat-treated milk.
- Treatment with 0.75% HPC aided MAP collection in the cream fraction.
- Heat treatment, pooling fractions, and HPC treatment significantly improved PCR detection.
- Washing pellets prior to DNA extraction did not enhance detection.
- The optimized procedure achieved a limit of detection of approximately 15-50 CFU/50 ml (
Conclusions:
- Pre-treatment methods, including heat treatment and HPC, are critical for efficient MAP recovery from raw milk.
- Optimized sample processing significantly enhances the sensitivity of PCR-based MAP detection in raw milk.
- The developed method offers a sensitive and reliable approach for detecting MAP in raw milk, with potential applications in food safety and disease control.
