PCR for food microbiology

Real-time PCR assays for food microbiology have been developed into commercial products whereby some or all of the steps can be automated to minimise the number of operations involved and reduce the risk of contamination. Automation requires a higher capital investment and so will depend on the sample throughput required. The reaction usually takes place inside a computer-controlled combined thermocycler/fluorescence detection instrument and uses pre-prepared reagents. For foodborne pathogen detection tests, the entire process can be completed within 20-30 hours with sufficient sensitivity to detect a single cell in a 25g sample.

The main advantage for PCR-based methods is in shorter detection times, but the high degree of automation built into PCR systems also allows relatively unskilled staff to run them without extensive training. The high specificity of PCR can also mean fewer repeat tests. The principal disadvantage is currently cost, both in terms of capital outlay and consumables. While larger laboratories can benefit from reduced labour costs, economies of scale and rapid results, the benefits for smaller labs may be less clear.

A number of commercial PCR systems are currently offered for food pathogen detection. One of the first into the marketplace was the Bax® system from Dupont Qualicon and this has been joined by TaqMan® and MicroSEQ® food pathogen detection kits from Life Technologies, iQ Check real-time PCR kits from Bio-Rad, foodproof® real-time PCR detection kits distributed by Merck, and several others.

One of the main reasons for the comparatively high capital cost of PCR-based detection systems is the need for thermocycling during the amplification step. Instruments must be capable of very accurate and precise temperature control throughout the cycle. In addition to the capital investment for the instrumentation, the chemistry is also expensive as it uses fluorescent probes. Novel developments in DNA synthesis have demonstrated alternative solutions for DNA amplification under isothermal conditions without the need for a thermocycler. There are several types of nucleic acid amplification technologies. Amongst the isothermal DNA amplification technologies that have been developed, loop-mediated isothermal amplification (LAMP) has been used widely to detect microorganisms and is a promising and suitable technology for the rapid detection of pathogen in the field. LAMP uses multiple primers and a bacterial polymerase, Bst polymerase, derived from Bacillus stereothermophilus to amplify DNA rapidly at a constant 63oC. This does away with the need for a thermocycler component in the instrument and can reduce the cost by up to two thirds.

A commercial isothermal amplification system has already been developed for food pathogen detection. The 3M™ Molecular Detection System uses a unique bioluminescence method to detect the amplification of DNA sequences and is designed to be simple to use. The amplification and detection processes are completed within 75 minutes with real-time positive results available as early as 15 minutes. An overnight single enrichment step is still required at present. Test kits for E. coli O157 including H7, Salmonella and Listeria spp. detection in food and environmental samples are currently available.

Portable PCR-based instruments have also been developed recently. For example, Idaho Technology markets the ‘Ruggedized Advanced Pathogen Identification Device’ or R.A.P.I.D.® System, which uses an air thermocycling process and a fluorimetric detection system to detect Salmonella, Listeria, E. coli O157 and Campylobacter in food samples.
Posted by Rapid Microbiology

PCR for detection and identification of carbapanem hydrolysis β-lactamases genes

A new paper of interest has been published, titled: “The current state of PCR approach in detection and identification of carbapanem hydrolysis β-lactamases genes”. The paper has been written by Tim Sandle, Dmitriy Babenko, Alena Lavrinenko, Ilya Azizov and Antonella Cheșcă (Pharmig, Karaganda State Medical University and University of Brașov).

Here is the abstract:

“Antibiotic resistance is arguably the most serious health-related issue of the current time. This is even more so with carbapenem-resistant Enterobacteriaceae, for such microorganisms are resistant to the carbapenems (the ‘antibiotics of last resort’). One of the most important considerations is in the detection of bacteria that carry the carbapenem-resistant gene. For this, molecular-based phenotypic and genetic-based polymerase chain reaction (PCR) methods are available. In contrast to phenotypic methods, molecular-genetic techniques, such as PCR, are considered to have the potential for improved detection of carbapenem-resistant genes by virtue of specificity, accuracy and rapidity. The tendency in PCR techniques is to develop towards the real-time systems equipped with multiplexing functionality. However, as shown in our study, standard PCR with electrophoresis detection continues widely to be used for the detection and identification of the carbapenemase gene. Therefore, despite progress in PCR technology, methods deployed for the detection of serious hospital acquired infections around the world are arguably neither the most accurate nor the most efficient. This issue is of concern for pharmaceutical scientists in relation to the use and development of PCR technology and in relation to new drug development.”

The reference is:

Sandle, T., Banenko, D., Lavrinenko, A., Azizov, I. and Chesca, A. (2014) The current state of PCR approach in detection and identification of carbapanem hydrolysis β-lactamases genes, European Journal of Parenteral and Pharmaceutical Sciences, 19 (1): 153-164

To obtain a copy, please contact Tim Sandle

Posted by Tim Sandle

PCR technique of genomic fragments from endogenous and adventitious viral agents

The European Medicines Agency have issued a document entitled “Detection by a highly sensitive new PCR technique of genomic fragments from endogenous and adventitious viral agents in live attenuated vaccines” (reference EMEA/H/A-5(3)/1269).

The document is concerned with the possible presence of nucleic acid sequences from endogenous and adventitious viral agents in live attenuated viral vaccines. The paper considers the potential of the analytical metagenomics approach (polymerase chain reaction (PCR) combined with pyrosequencing, for the detection of viral fragments.

In summary, the statement outlines the following position:

1. Whilst the presence of endogenous ALV and SRV DNA fragments is potentially linked to the cell lines used for the manufacture of the individual vaccines, the finding of PCV-1 DNA pointed towards the presence of non-endogenous viral DNA. The Committee is therefore asked to assess if there is any potential public health concern arising from all the findings described in the article.

2. It is understood that as more hi-tech analytical methods become available, new findings may reveal in medicinal products presence of substances at very low concentrations and/or hard to detect that were thus far not picked up because of limitations of the analytical instruments/methods declared and authorised in the marketing authorisation dossier. Specifically, the Committee is asked to indicate if the new test method described above applied to identify viral genomic nucleic acid fragments should be considered, if validated, for the development/qualification and/or routine testing of biological medicinal products, and particularly live attenuated vaccines. Recognising the limitations of this novel method, and in order to conclude on the presence or absence of specific risks, the Committee is also asked to consider whether additional tests would be needed to conclude on the presence of replication competent endogenous and adventitious viral agents.

3. The Committee should indicate if there is a need to update existing guidance related to the testing and elimination of such substances in the context of development and/or testing of live attenuated vaccines.

4. Depending on the outcome of these investigations, the Committee should consider the need for appropriate guidance for other vaccines and other biological products.

The statement can be found at: EMA

Posted by Tim Sandle