The Rapid Micro Blog

Our blog will keep you informed of new and noteworthy technologies, reviews of recent publications and presentations, upcoming conferences and training events, and what's changing in the rapid and alternative microbiological methods world.

HPLC of PCR Products Now Being Used to Detect Pathogens in Food

Recent developments in rapid methods have led researchers to utilize multiplex polymerase chain reaction (PCR) and high-performance liquid chromatography (HPLC) for the identification of food borne pathogens, such as diarrheagenic E. coli. A recent overview from discusses this new application, and I have provided an excerpt below.

Dangers of Diarrhoea

Children are at higher risk of contracting diarrhoea than adults due to their underdeveloped immune systems and they are likely to be affected for longer periods. In the Western world, most cases are easily treated but it is a different story in developing countries, where infection rates are higher and death is a common outcome.

Acute diarrhoea is generally caused by bacterial, viral, or parasitic infection and one of the key bacteria is diarrhoeagenic Escherichia coli, which can enter the body via contaminated food. However, it is not simply one strain that is responsible. In foods, four main categories can cause diarrhoea: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorraghic (EHEC) and enteroinvasive (EIEC) E. coli.

Each of these categories contains similar but individual DNA, which proved a blessing for a team of Chinese scientists who have exploited the differences in a novel detection method. Lichun Cui from the Northeast Forestry University, Harbin, with colleagues from the Northeast Agricultural University, Harbin, and the Heilongjiang and Hainan Entry-Exit Inspection and Quarantine Bureaus, devised a procedure based on denaturing HPLC that could detect single or mixed E. coli infections in food.

Denaturing HPLC

In denaturing HPLC, mismatches in the DNA bases of double-stranded DNA allow their separation provided certain criteria are met. At elevated temperatures, the hetero and homo duplex chains unwind, or denature, to their individual strands which are resolved on the HPLC column.

The HPLC stationary phase must be inert and electrically neutral. Under these conditions, DNA cannot bind due to its inherent negative charge but the addition of an ion pairing agent to the mobile phase changes the properties and binding is achieved via electrostatic interactions.

The hetero duplexes are denatured to a greater extent than the homo duplexes, so that they are retained less strongly on the column and elute first. So, pairs of hetero and homo chains are observed in the HPLC chromatograms.

In the case of E. coli, the researchers employed a poly(styrene-divinylbenzene) column and added triethylammonium acetate to the mobile phase for ion pairing. Separation was effected with a gradient of acetonitrile.

DNA was extracted from E. coli bacteria and subjected to polymerase chain reaction (PCR) amplification using unique primers based on the conserved regions of each of the four bacteria. Sufficient sample was generated for HPLC analysis and only the expected products were produced, as proven by agarose gel electrophoresis. The average size of the products was 220, 300, 330 and 500 base pairs for ETEC, EPEC, EIEC and EHEC, respectively.

Bacterial Strains Detected Together in Food

The HPLC separations were carried out at 50°C under non-denaturing conditions, which produced a single peak for each amplified fragment. They eluted at different retention times over 4-9 minutes, allowing them to be distinguished from each other.

The PCR products from all four E. coli categories were then mixed together for HPLC, confirming that they can be separated and distinguished using their retention times.

The critical step in the process is the specificity towards each category. This was assured by subjecting the genomic DNA from 34 bacterial strains to the same amplification and analysis process using the unique primers. Only the ETEC, EPEC, EIEC and EHEC strains and their isolates gave positive results.

The novel procedure was used to test 189 samples of faecal matter from patients as well as 690 import and export food samples, including beef, pork, chicken, sausages and milk. Blind testing was carried out and the results were compared with those from the conventional method which involves bacterial culture over 2-3 days and biochemical reactions.

A total of 60 positive samples were identified with all four strains being detected. The results were in perfect agreement with the conventional method, confirming the validity of the approach. However, the PCR denaturing HPLC method is much faster and provides a valid alternative. It could also replace the basic PCR assay which requires a gel electrophoresis step.

The multiplex method is capable of detecting all four strains in one procedure, so can identify one or more of the infections in contaminated food and in patient faeces, permitting rapid detection and diagnosis and reducing the time before appropriate action can be taken.

The original article appears here. Image by Renjith Krishnan.


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