How microbial genetics can be used in the identification of different Bacteria
Since the discovery of DNA, it becomes a useful diagnostic tool. In cancer studies, species identification, diagnosis of inherited disease and in other research field different types of genetic tools are routinely used.
Microbial genetics gained substantial attention in recent years. Microbiology is one of the traditional fields of science which deals with microorganism and their related enforcements. Even it is a standard tool in the diagnosis of disease.
If you want to study a group of some specific type of bacteria, sequence specific primers (which are now commercially available) are used to amplify DNA of different bacteria. Based on the data of the size of amplicon, we can identify different bacteria.
Though microbiology is a vast field and it is well established in all scientific areas, use of microbiological tool has several limitations.
The first and foremost limitation is a time of culture.
For identification of any microbe, for example, any bacteria, it is important to culture that bacteria on some growth medium and it will take minimum 24 – 48 hours to grow. Even after culture, there is no surety of result.
another factor which hinders in culture is a chance of contamination. Plating or culturing is one of the sensitive methods in biological sciences because the chance of contamination is always very high in culturing.
In simple words, culturing is a technique in which any type of unknown bacterial sample can be cultured or grown on a nutritional agar plate. The agar plate contains all the essential nutrients like proteins and vitamins needed for bacteria.
Thousands of different bacteria are lived in the air, on our hand, in our breath or even on the bench on which we are working. So failure is a strong possibility is culturing because of the risk of contamination.
If the culture is contaminated, a lot of different types of bacteria will grow in a culture plate hence contamination is a major limitation of culture technique.
Even it takes more time for proper growth so in case of diagnosis of life-threatening disorders patient may die.
The microscopic analysis is another factor which pushes back the traditional microbiological culturing method. The chance of error is always very high in case of microscopy. Furthermore, the wrong interpretation is one of the strongest possibility of a result.
If we want to identify more than one type of bacteria at once, the culture method becomes more tedious. Hence the use of microbial culture technique remains always controversial in science.
In recent years, microbial genetics provides an opportunity to the scientist in which genetic information of any microorganism used as a tool in research and diagnosis. Several sequences are present in all bacteria with slight variation, can be used in the identification of different bacteria.
16s rRNA is a type of RNA sequence which can transcribe into rRNA.16s is a sub unit of a ribosome and the sequence is ribosomal RNA.
16S rRNA sequences are a unique type of homologous sequences which are similar in all bacteria and Archaea. It contains hypervariable regions (V1-V9). These regions are variable among the species and within the species.
Within the species, variability in a hypervariable region is lower and among different species, the variability is higher. However, it contains some homologous sequences which are similar in all prokaryotes.
Universal primers are designed based on the data of sequence homology and it is used for identification of all types of prokaryotes specifically bacteria and archaea.
DNA is isolated from a bacterial sample and 16s rRNA gene is amplified using universal 16s rRNA primers. Sanger sequencing helps in identification of sequence variation. With the help of BLAST-like bioinformatic tools, sequence homology is checked with different bacterial sequences.
Within a few hours, we can obtain the results. The technique is simple to perform and the chance of cross contamination is fewer. Even if only a few or single bacterium is present in the sample, the molecular technique gives the proper result.
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Furthermore, it is very difficult for counting numbers of bacteria into the sample or the load of bacterial infection in sample with the help of microbiological techniques.
However, By using RT PCR (realtime PCR), we can count the number of bacteria present in the sample, based on the number of times 16S rRNA gene amplified.
Sequence-specific primers are now available which amplifies DNA sequences of one specific bacteria. Sequence-specific primer of TB bacteria, can amplify sequences on TB bacteria from the sample, even if some other bacteria are present in the sample, it can only amplify TB bacterial gene.
It is rapid and gives result in hours. So molecular genetic techniques play crucial role in microbial identification and disease diagnosis.
Yet another drawback of microbial culturing is, investigator could accidentally be infected with unknown sample.
Here molecular genetic technique gives additional benefit in which chance of infection is very less. Once an investigator isolated DNA from bacteria, it is totally safe to handle it in the free environment.
Can you identify different type of bacteria from a single sample with the help of platting?, well, it is quite tedious. Suppose you have a urine sample and you want to analyze the type of bacteria in urine, it is difficult to identify all type of bacteria from platting.
Interestingly, metagenomic analysis helps to do the work.
In metagenomics analyses, an investigator can identify multiple type of bacteria from an unknown sample with the help of molecular genetic techniques. Feces sample, urine sample, soil sample and other environmental samples are routinely analyzed using metagenomics.
If you want to study a group of some specific type of bacteria, sequence specific primers (which are now commercially available) are used to amplify DNA of different bacteria. Based on the data of the size of amplicon, we can identify different bacteria.
Even, for identification of unknown bacterial strain 16S rRNA helps to investigate new strains. If the sample contains multiple types of bacteria or multiple strains of bacteria, a different amplicon of 16S rRNA are sequenced and with the help of bioinformatic tools bacterial strains or the different type of unknown bacteria can be identified.
Molecular genetic techniques are reliable, accurate, advance, rapid, safe and faster than traditional microbiological platting and culturing. Advancement in microbial genetics gives a new direction to the microbial research.
Article story written and covered by – Tushar Chauhan
Article reviewed by – Tushar Kachhadiya
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