Clear detection of pathogenic microorganism: no more Russian roulette
How can molecular biology help to identify viruses and bacteria?
This year the corona virus appears as an aggressive type of virus. Other viruses, such as the Norovius, are also diagnosed as threatening germs. The major foodborne illnesses are caused by bacteria and viruses. Specific bacteria can cause severe infections of the stomach and intestine (acute gastroenteritis) and can become life threatening.
For instance, some time ago in December a cruise ship was put under quarantine after at least 70 passengers fell sick with vomiting and diarrhoea. In such cases a fast sensitive diagnostic method is needed in order to treat diarrhoea and to release the victims from quarantine. Within a few days, the norovirus was identified as the cause of the gastroenteritis outbreak and the victims could be treated accordingly.
The norovirus indeed is one of the most infectious viruses in humans and is very prevalent, also beyond this instance on a cruise ship that received much media attention. The norovirus was also responsible for the biggest foodborne disease apparent in many day care centres and schools in eastern Germany. In 2012 over 11,000 children became ill within a few days, again with vomiting and diarrhoea. Even though also here the norovirus was identified quickly as the cause, it took much longer to identify its source, deep frozen strawberries imported from east asia, indicating how quickly the virus can spread across the world.
Meanwhile we know that such symptoms of acute gastroenteritis, where diarrhoea is combined with vomiting, can also be caused by bacteria. In summer 2011 a variant of the enterohaemorrhagic Escherichia coli (EHEC) caused a serious outbreak of foodborne illness concentrated in northern Germany. The sickness is characterised by serious complications including bloody diarrhoea and a haemolytic-uremic syndrome (HUS). Because of the acute renal failure, in such cases an urgent treatment is required. In June 2011, the Federal Institute for Risk Assessment (BfR), the Federal Office of Consumer Protection and Food Safety (BVL) and the Robert Koch Institute (RKI) identified that fresh vegetables were most likely infected with EHEC bacteria. Yet, after intensive investigations the initial warning for consumption of raw salad, tomatoes and cucumbers was withdrawn. Later it turned out that the most likely sources of the contaminated food, were certain types of spouted seeds from Egypt. At the end of June 2011 the Robert Koch Institute declared the end of the EHEC outbreak. During this EHEC epidemic, 53 people died and 3,842 became ill, some of them are still seriously affected.
There is currently no simple protection against diarrhoea caused by viruses or bacteria
Recently, researchers from Ohio State University showed that common cleaning methods are not very effective against some of the microorganisms. They showed, for instance, that the norovirus can withstand both manual and mechanical washing.
The researchers contaminated cream cheese and reduced-fat milk with a norovirus, with E. coli or with Listeria, three common causes of foodborne illnesses. These dairy products were then applied to stainless-steel silverware, ceramic plates and drinking glasses. The utensils were washed by hand or in a dishwasher following sanitizing protocols that used chlorine and quaternary ammonium compounds. Both hand-washing and dishwashers reduced E. coli and Listeria to safe levels. However, the researchers found neither cleaning method could reduce the presence of the norovirus. This indicates that more care needs to be taken, and that better agents and methods need to be identified, to significantly prevent cross-contamination of food and dishes at food-service establishments.
Detection of harmful organisms with Real-Time PCR
There are billions of bacteria and viruses in our environment. Most of them are harmless and live in the ground, in the water, on plants and animals and also in our intestinal tract. In our gut the bacteria are essential and thus friendly helpers. They help us to digest food and to absorb nutrients. But how can we recognize and differentiate between the good bacteria and the harmful bacteria. Many of the harmful bacteria are variants of the good bacteria and have acquired pathogenic characteristics. For example the EHEC bacteria is a closely related strain of the E coli bacteria but gained pathogenic capacities with the ability to produce harmful toxins.
The best and efficient method for the detection and identification of a specific pathogenic organism or an organism group is Real-Time PCR. The technique is based on the exponential amplification of DNA by thermostable Thermaus aquaticus (Taq) polymerase. DNA isolated from samples can then be directly analysed. When virus-RNA is used, an enzymatic treatment of reverse transcriptase reaction is needed to reverse transcribe RNA into DNA. The analysis and quantification of DNA or RNA is based on a fluorescence kinetic PCR and enables the quantification of the PCR product in real time. This sensitive and accurate technique permits quantification of PCR products during the exponential phase of PCR reaction and provides information as rapidly as the amplification itself. The development of this real time PCR had a revolutionary impact on molecular diagnostics.
Biobioseminars (www.biobioseminars.com), at the interface between biology and medicine, offers you seminars and practical courses in Real-time PCR and its application for rapid diagnostics.
Virus, virus infection, virus detection, Virus Real-Time PCR, virus quantitative PCR, qPCR, detection of pathogenic microorganisms, foodborne illnesses, diarrhoea, bacteria, viruses, norovirus, enterohaemorrhagic Escherichia coli (EHEC), exponential amplification of DNA, quantification of the PCR product, Biobioseminars, rapid diagnostics.
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