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Methods Used to Detect Enteric Pathogens

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  • OMOYOSOLA EKOSANMI

Antibody & DNA Technology

Compare and contrast Immunological and Molecular methods used to detect Enteric Pathogens

Enteric pathogens are gram-negative organisms that live and are found in the human gastrointestinal tract, some examples of enteric pathogens are Clostidium difficile, Campylobacter, Bacillus cereus and Salmonella. Most enteric pathogens that enter the gut won’t cause any harm but there are some harmful bacteria that do cause infection in the stomach called gastroenteritis. There are a number of molecular and immunological methods scientifically used to detect and distinguish different enteric pathogens such as PCR and ELISA. It is important to be able to detect enteric pathogens correctly to make sure that they are treated effectively, ‘accurate identification of these pathogens plays a big role in microbiology and infectious diseases’ (Janda and Abbott, 2002)This essay will look at and discuss the similarities and differences of the different methods by comparing and contrasting.

Molecular methods are much more convenient and advanced than conventional methods being that they are quick and sensitive as opposed to immunological methods since they can only detect a certain amount of pathogens at a time and also they are not as sensitive and specific. (Boer et al, 2010) It is important to use the correct method when detecting different pathogens to ensure consistent results. Molecular methods can be used to detect various types of enteric pathogens some of methods includes Polymerase Chain Reaction (PCR) that is commonly used technique, it is much more advanced in detecting pathogens such as Salmonella, Shigella and E. coli O157 and even the causes of diarrhoea compared to cell cultures, PCR has lots of benefits such as it is very specific, speedy, fairly cheap however it is time consuming an assay can take quite a long time to produce results however PCR is fairly easy to learn (Abbaszadegan,2004). A standard PCR would not be able to detect a large number of pathogens, and is restricted in the number of samples it can detect at once which is similar to western blotting (Jun-wen et al, 2004) unlike ELISA which is capable of detecting lots of samples . Real time PCR is more advanced than a standard PCR because it allows us to know exactly how much a DNA has amplified and can be used to detect pathogens like Clostidium difficil, the real time PCR like the standard PCR is specific and very sensitive and doesn’t not require a lot of time likewise to the EIA technique it is not time consuming however it is not as reliable because it is not as sensitive (Bélanger et al,2003). To detect the present of toxin b Clostidium difficil samples a method called tissue cytotoxic neutralization assay can be used similar to the PCR method, it takes around 48 hours to produce results unlike real time PCR and EIA which can produce results in an hour (Bélanger et al,2003).

Fluorescence in situ hybridization (FISH) technique is used to locate and identify DNA sequences on chromosomes and to detect chromosome defects, it can be used to examine and locate the the bacteria pathogens in human intestines like Enterococcusspp, this technique is simple and effective just like. The FISH technique is sensitive but this due to how the technique is carried out (Jonach et al, 2014). DNA hybridisation looks at two strands of DNA and see how they are alike, this method can be compared to PCR because needs the ’DNA tested needs to be in large quantities’ (Goris et al, 2007). E.Coli is a common enteric pathogen that is detected using DNA hybridisation and it is very effective, this technique is very specific which is the same as the ELISA technique. DNA sequencing is an effective molecular technique which examine parts of a DNA, DNA sequencing can be used to detect the genes associated with a certain bacteria

Immunological methods used in the detection of enteric pathogens mainly reply on the antigen-antibody reaction, immunological methods are able to detect the immunoglobin that are specific to the pathogen (Siqueira and Rocas, 2005). However immunological techniques are known to be time-consuming unlike the new molecular techniques like real time PCR, but most immunological techniques are very sensitive and specific just like most molecular techniques but they are limited in how many pathogens they can detect. Immunofluorescence assay (IFA) this method identifies antibody and antigen binding by the use of fluorescence, it can detect pathogens like Salmonella and Cryptosporidium. IFA technique is not as reliable which differs from immunological techniques like rapid agglutination assays and ELISA, also a major difference between IFA and ELISA is that the person operating the IFA will need to do most work because the IFA depends mostly on the person carrying out the assay (Bayer et al, 2001). On the other hand both IFA and PCR are very sensitive and specific (Russel et al, 2013). Just like the PCR technique IFA offers high sensitivity and it is specific(Ahmed and Chowdhary, 2013)

Enzyme linked immunosorbent assay (ELISA) a immunological technique used to detect antibodies and also observes the way they change colour in response to identification of a substance, a colourless substrate reacts with a given enzyme this will then result in a colour change. This means that when a coloured product is shown an antigen is present. ELISA is very effective in detecting pathogens such as Rotavirus, Adenovirus and Campylobacter. The ELISA method is quick (results can be produced in 24 hours), precise and very convenient it can. This technique is similar and can be compared to PCR because of its rapidness and sensitivity. ‘The PCR method is 100-1000 more sensitive than traditional cell culture based methods’ (Lui,2009) but can give false results

Monoclonal and polyclonal antibodies can be used for the ELISA method. The ELISA can also be used to find out which antigens produce some of the harmful pathogens for example Campylobacter, so the Campylobacter pathogen does not need to grow a lot and the ELISA test does not rely on its growth (Jeremy et al,2002).

Enteric pathogen can be identified based of approaches such as immunohistochemical detection which can be used to identify pathogens like Helicobacter pylori , however these kinds of techniques are restricted on how many pathogens they can detect at once which is similar to a standard PCR. Advantages of immunohistochemical is that it is quick, sensitive and safe to use just like the FISH technique. Mac-Conkey agar is a seletive media used to isolate and identfy enteric gram negative bacteria that fermate lactose , they are quite cheap to run and very effective in deteting. (Acharya, 2013). Reversed passive latex agglutination (RPLA) is also an immunological method it is fairly cheap, speedy and easily to carry out which can be compared to PCR. Western blot another immunological technique involves identifying certain proteins in a sample both ELISA and western blot are both used to detect proteins, western blot are sensitive however they are require a lot of time, they are not simple to run and it is restricted in the number of samples it can process which can be compared to IFA, it would need someone which a bit more experience to operate it unlike the PCR which is a simple procedure (Grys et al, 2009). A major difference of Enzyme immunoassays in contrast to PCR is not as sensitive and specific EIA sensitivity (Belanger et al,2003), PCR is able to detect more specimens when tested and compared against EIA, EIA was slower at detecting specimens as opposed to PCR which is a rapid and quick test (Grys et al, 2009). Rapid agglutination assays is an immunological method that can be used to detect pathogens for example it is commonly used in for Salmonella, this test is differs from PCR because it is not as specific however it is a simple, quick reliable test making it more convenient to use than conventional methods

To conclude both molecular and immunological techniques plays a huge role in identifying and treating pathogens, molecular techniques are much more convenient than immunological techniques on the other hand immunological techniques are very useful when detecting certain pathogens. PCR is one of the most used and convenient method for detecting pathogens and has many benefits than any other method. Most of the methods are very quick to do and can produce results in less than 24 hours. New advanced methods are the most common used by scientists because of its sensitivity, specificity and rapid results. Most immunological methods are useful but may not be used as much as molecular methods because of their lack of specificity and complex use. Enteric pathogens like E.Coli 0157, Salmonella and Shigella are the most common so it is very important to be able to detect their pathogens effectively using the correct methods.

References

Acharya T. (2013). MacConkey Agar (MAC): Composition, preparation, uses and colony characteristics.Microbonline - Online Medical Microbiology Guide. 2 (1), 2-4.

Ahmed NH, Chowdhary A. (2013). Comparison of Different Methods of Detection of Enteric Pathogenic Protozoa.Indian Journal of Medical Microbiology. 31 (2), 154-160

Bayer PM, Fabian B, Hübl W. (2001). Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics – technique, benefits, limitations and applications .Scandinavian Journal of Clinical & Laboratory Investigation. 61 (235), 68-78.

Bélanger SD, Boissinot M, and Bergeron MG. (2003). Rapid Detection of Clostridium difficile in Feces by Real-Time PCR.Journal of Clinical Microbiology. 41 (2), 730-734.

Boer RF, Ott A, Kesztyüs B and Kooistra-Smid AMD. (2010). Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach.Journal of Clinical Microbiology. 48 (11), 4140-4146.

Grys TE, Sloan LM, Rosenblatt JE, Patel R.. (2009). Clinical Microbiology - Pubmed .Rapid and sensitive detection of Shiga toxin-producing Escherichia coli from nonenriched stool specimens by real-time PCR in comparison to enzyme immunoassay and culture.. 47 (7), 2008-2012.

Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vanamme P and Tiedje JM. (2007). DNA–DNA hybridization values and their relationship to whole-genome sequence similarities.International Journal Of Systematic And Evaluatorary Microbiology. 57 (1), 81-91.

Janda JM and Abbott SL. (2002). Bacterial Identification for Publication: When Is Enough Enough?.Journal of Clinical Microbiology. 40 (6), 1887-1891.

Jeremy M Berg, John L Tymoczko, and Lubert Stryer. (2002).Biochemistry. 5th ed. New York : W H Freeman. p567-580.

Jonach B, Boye M, Stockmarr A and Jensen TK. (2014). Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea.BMC Veterinary Research. 10 (68), 4-8.

Liu D (2009).Molecular Detection of Foodborne Pathogens. USA: Taylor and Francis group . p27-28.

Morteza Abbaszadegan. (2004). Civil and Environmental.Microbial Detection Methodologies. 18 (1), p19-35

Russell H, Sampson JS, Schmid GP, Wilkinson HW and Plikaytis B. (2013). Enzyme-Linked Immunosorbent Assay and Indirect Immunofluorescence Assay for Lyme Disease.The Journal of Infectious Diseases. 149 (3), 465-470.

Siqueira J.F and Rôças I.N. (2005). Exploiting Molecular Methods to Explore Endodontic Infections: Part 1—Current Molecular Technologies for Microbiological Diagnosis.Journal of Endodontics. 31 (6), 411-420.

Wen J, Shi XQ, Chan FH, Wang XW, Zheng JL, And Song N . (2004). A Study on Detecting and Identifying Enteric Pathogens With PCR.BIOMEDICAL AND ENVIRONMENTAL SCIENCES. 17 (2), 109-120


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