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Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
A comparison of DNA extraction and purification methods to detect Escherichia coli O157:H7 in cattle manure
Teegan Trochimchuk1, John Fotheringham, Edward Topp
1Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1.
Abstract:
The extraction of DNA from manure and the subsequent polymerase chain reaction (PCR) amplification of virulence genes to detect pathogens require an effective method of purification. Four different methods were assessed for their effectiveness in extracting and purifying Escherichia coli O157:H7 DNA from cattle manure: phenol/chloroform purification, phenol/chloroform/Sepharose B4 spin columns, phenol/chloroform/polyvinylpolypyrrolidone (PVPP) spun columns, and Mo Bio UltraClean kit. A PCR assay targeting the shiga-like toxin I gene (sltI) was carried out to determine the effectiveness of the four methods in removing PCR inhibitors from the manure samples. All methods were used to extract a manure slurry and the cleanliness of the samples was tested by the PCR with varying concentrations of spiked E. coli O157:H7 target DNA. The PVPP spun columns and the UltraClean kit had the best detection limit, detecting 20 pg of E. coli DNA (about 2x10(3) cells) per 100 mg of manure. The UltraClean kit and the PVPP spun columns also had the best and similar detection limits of 3x10(4) CFU/100 mg manure when E. coli O157:H7 cells were spiked into the manure sample and purified by all four methods. The enrichment of cells after inoculation into manure was performed using tryptic soy broth at 37 degrees C for 5 h. Both the PVPP spun columns and the UltraClean kit methods were used to purify the enriched samples and were able to detect initial inocula of 6 CFU/100 mg manure, indicating that the two methods were highly efficient in purifying DNA from manure samples.
Insights
This study compared DNA purification methods for detecting Escherichia coli O157:H7 in cattle manure using polymerase chain reaction (PCR). The polyvinylpolypyrrolidone (PVPP) spun columns and Mo Bio UltraClean kit demonstrated superior DNA extraction and purification efficiency.
Area of Science:
- Environmental microbiology
- Molecular diagnostics
- Bacterial pathogen detection
Background:
- Effective DNA extraction and purification from complex matrices like manure is crucial for pathogen detection.
- Polymerase chain reaction (PCR) is a sensitive method for amplifying specific DNA sequences, but inhibitors in environmental samples can hinder its performance.
Purpose of the Study:
- To evaluate and compare four different DNA purification methods for extracting and purifying Escherichia coli O157:H7 DNA from cattle manure.
- To determine the efficiency of these methods in removing PCR inhibitors present in manure samples.
Main Methods:
- Four DNA purification methods were assessed: phenol/chloroform, phenol/chloroform with Sepharose B4 spin columns, phenol/chloroform with polyvinylpolypyrrolidone (PVPP) spun columns, and the Mo Bio UltraClean kit.
- A PCR assay targeting the shiga-like toxin I gene (sltI) was used to assess the purity and detectability of extracted DNA from spiked manure samples.
Main Results:
- The PVPP spun columns and the Mo Bio UltraClean kit exhibited the best detection limits, identifying as little as 20 pg of E. coli DNA (approximately 2x10^3 cells) per 100 mg of manure.
- Both methods also showed superior performance with spiked E. coli O157:H7 cells, achieving detection limits of 3x10^4 CFU/100 mg manure.
- After enrichment, these two methods successfully detected initial inocula as low as 6 CFU/100 mg manure, indicating high purification efficiency.
Conclusions:
- The PVPP spun columns and the Mo Bio UltraClean kit are highly effective for purifying DNA from cattle manure for pathogen detection.
- These methods significantly reduce PCR inhibitors, enabling sensitive detection of Escherichia coli O157:H7.
