Using Pcr To Identify Foodborne Pathogens In Future Biology Essay

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The World Health Organisation (WHO) defines food-borne illnesses as diseases, usually either infectious or toxic in nature, caused by agents that enter the body through the ingestion of food. "Though the global incidence of food-borne disease is difficult to estimate, it has been reported that in 2005 alone 1.8 million people died from diarrhoeal diseases and a great proportion of these cases can be attributed to contamination of food and drinking water" (WHO, 2010).

"Food-borne disease is a major cause of illness in the UK population and imposes a significant burden on patients and the economy. It is estimated that in the UK in 2007 around a million people suffered a food-borne illness, which led to 20,000 receiving hospital treatment and 500 deaths, at a total annual cost of nearly £1.5 billion" (Food standard agency, 2010). Several steps have been taken at the legislative levels to improve food safety. Nevertheless, due to the increase of travel and trade, food-borne diseases can spread fast rapidly inflicting major effect on health and economy. To prevent food-borne infections it is essential to detect the pathogen responsible, rapidly and reliably. Traditional culture methods for the detection of food pathogens are time consuming, requiring several days to acquire positive results (Myers et al., 2003). One of the natural problems in the detection of food pathogens is that they are present in small numbers, which may results in them being lost among normal microflora, other pathogenic micro-organism, or foodstuff itself. (Naravaneni and Jamil, 2005).

To confront these problems gene probe techniques were introduced. These techniques have allowed the development of powerful tests by which specific bacterial strains can be quickly identified without the need for isolating pure cultures. Researchers have developed a PCR-based assay for the detection of food-borne pathogens. In this essay I will consider the contribution of the Polymerase Chain Reaction (PCR) to the detection and characterisation of food-borne pathogens.

PCR is finding an increasing application as a specific and sensitive method for the detection of food-borne pathogens (Bhaduri et al). Many papers have been published explaining the development different PCR methods that can be adapted to detect different bacteria. "PCR techniques have significantly improved the detection and identification of bacterial pathogens" (Nogva, H. K., 2000). This explosive increase since the beginning of the 1990s in the number of publication reporting PCR-based methods for detection of food-borne pathogens has allowed for more reliable microbial identification and observation. PCR has also become a valuable tool for investigating food-borne outbreaks and identifying the responsible etiological agents. The PCR method have provided amplified sensitivity, allowed for more fast processing times, cheap and enhanced the possibility of detecting bacterial pathogens. In addition to the investigation of foods, PCR has also been successfully applied to the detection and identification of pathogenic organisms in clinical and environmental samples (Lampel K.A., 2000).

PCR is a technique for in vitro amplification of specific segments of DNA by using a pair of primers (R. Naravaneni and Kaiser Jamil, 2005). PCR enabled scientists to amplify genes specific to the pathogenic food-borne bacteria and also to detect genes involved in the virulence of food-borne bacteria (Finlay & Falkow, 1988; Bej et al, 1994). This newly developed technique has allowed the detection of small amount of target DNA by amplifying specific DNA sequences. By amplifying a gene that is specific to the pathogenic micro-organism of interest, PCR can be used to ultimately detect very low number of microbes (Naravaneni and Jamil, 2005).

One major food-borne pathogen is the diarrheagenic E. coli which represent a major health concern in developing countries (nataro et al., 1998). The introduction of PCR has made it possible to detect and identify all strain of diarrhegeanic E.coli in one single reaction tube using multiplex PCR, bearing in mind that PCR methods using one single primer sets to identify individual strains of this organisms have been reported in previous studied. Toma et al. 2003 developed a multiplex PCR assay for the identification of human diarrheagenic Escherichia coli. The following genes for each serotype of E. Coli O 157:7h; eae for enteropathogenic E. Coli (EPEC) , stx for Shiga toxin-producing E. Coli (STEC), elt and est for enterotoxigenic E. Coli (ETEC), ipaH for enteroinvasive E. coli (EIEC), and aggR for enteroaggregative E. Coli (EAEC). Once theses specific genes were amplified using the designated primer sets, they compared their results to that of single PCR amplification, and concluded that they were able to identify various subgroups of different strains of the above organism. They also concluded that multiplex PCR "is a practical and rapid diagnostic tool for identification of diarrheagenic E. coli in a single reaction tube".

Lambertz et al., designed a PCR assay to amplify a sequence on the gene ail located on the chromosome of Yersinia spp. Associated with pathogenicity in humans and thus indicates the presence of the organism and defines the pathogenic subgroup at the same time. They developed a real-time PCR method to restrict the detection method to only the Yersinia enterocolitica only. This method will only amplify the ail gene on the Y. Enterocolitica only compared to the conventional PCR which will use primer sites on the ail gene that are present on both Y. Enterocolitica and Yersinia pseudotuberclosis. Their results showed that the primers selected provided specific detection of a large number of pathogenic Y. Enterocolitica strains isolated from the infected patents, animals, foods, and unknown sources (Lambrtz et al., 2008).

Another example is the detection of salmonella in clinical sample target the invA gene. The invA gene codes for protein in inner membrane of bacteria, which is necessary for invasion to epithelium cells. Salmonella specific PCR with primers for invA is rapid, sensitive, and specific for detection of Salmonella in many clinical samples (Lampel et al., 2000). Primers and probes where designed to target the invA gene in the salmonella. When isolates screened by PCR, it resulted in 248 bp amplified fragments, and No amplified DNA fragments were obtained from non-Salmonella species. The ability of Salmonella specific primers to detect Salmonella species rapidly and accurately is primarily due to the primer sequences that are selected from the gene invA of S. Typhimurium. Other studied gave supported the ability of these specific primers sets to confirm the isolates as salmonella (salhi et al. 2005). Other researchers have tried to establish methods which reduce the period of time in which salmonella strain can be detected. Guo et al. (1999) used primer set invA/invE for confirmation of isolated Salmonella from turkeys. Ferretti et al. (2001) proposed a rapid method with primers salm 4 and salm for invA gene, which allows the detection of Salmonella serotypes within a maximum of 12 hrs. Schneder et al. (2002) design a lightcycler Salmonella detection kit that enables the user to detect one single colony forming unit Salmonella in 25g sample in less than 24 hrs.

The above examples supported by many other publications support the advantages of PCR over other conventional methods. High specificity, selectivity, rapidity, sensitivity are among the important advantage of PCR detection methods (Malorny et al., 2003). Although conventional culture methods remain the most used and most reliable technique for the detection and identification of food-borne pathogens, the drawbacks are of major concern in terms of rapidity. Another drawback is the laborious procedure of culture methods as well as time consumed to carry out these methods. Clinical scientist spend a lot of time and effort to carry out the detection process, confirmatory results are obtained after at least a couple of days of culturing the organism, Whereas, PCR can take a few hours maximum to achieve a positive detection. Nevertheless, PCR has its disadvantages as well; it relies on the purity of the target template and presence of sufficient numbers of target molecules (Lampel et al., 2000). PCR can also be hindered by various inhibitors present is samples, foodstuff.

In conclusion, reviewing a number of literatures I have gathered that PCR approach is the strongest in terms of detection and identification of food-borne pathogens compared to that of traditional culture methods and other molecular methods. Despite its drawbacks PCR has the highest sensitivity and specificity. Using PCR one is able to selectively amplify a fragment of DNA that is specific to a particular species or strains.