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An efficient DNA extraction method for polymerase chain reaction-based detection of Mycobacterium avium subspecies
Michael Z Zhang1, Shuping Zhang
1Mississippi Veterinary Research and Diagnostic Laboratory, Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Pearl, MS, USA.
Abstract:
Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 × 10(7) CFU per ml or CFU per g). The detection limit of the real-time PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the "gold standard," the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.
Insights
A new DNA extraction method improves the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in cattle feces. This efficient method enhances the sensitivity of polymerase chain reaction (PCR) tests for diagnosing Johne's disease.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Bovine Diseases
Background:
- Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle is challenging to detect due to its lipid-rich cell wall and intermittent shedding in feces.
- Existing DNA extraction methods often yield insufficient high-quality MAP DNA, compromising the sensitivity of PCR-based diagnostic tests.
Purpose of the Study:
- To develop and evaluate an efficient DNA extraction method for improved PCR-based detection of MAP in bovine fecal samples.
- To optimize DNA yield and purity for enhanced diagnostic accuracy in Johne's disease surveillance.
Main Methods:
- Development of the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, incorporating chemical pretreatment, 1-tube lysis and extraction, chelex absorption, and mini-column purification.
- Quantification of DNA yield and purity using spectrophotometry (3.36 fg/CFU, A260/280 ratio of 2).
- Evaluation of the method's performance using real-time PCR and validation through Johne's direct fecal PCR proficiency tests.
Main Results:
- The MVRDL method demonstrated a linear correlation between cycle threshold (Ct) values and log input CFU, with a detection limit of 3 CFU/ml or CFU/g.
- Validation against culture results showed 100% specificity and 98.46% sensitivity for MAP detection.
- The method achieved a DNA yield of 3.36 fg per CFU with an A260/280 ratio of 2, indicating high purity.
Conclusions:
- The MVRDL DNA extraction method is highly efficient and superior to previously published protocols for MAP detection in bovine feces.
- This optimized method significantly improves the sensitivity and accuracy of PCR-based diagnostics for Mycobacterium avium subspecies paratuberculosis in cattle.
- The MVRDL method offers a valuable tool for Johne's disease surveillance and control programs in the veterinary field.
