Microbial Forensics

Microbial Forensics

The anthrax bioterrorism scare in 2001 that followed 9/11 Twin Towers  attack  further  decreased  America’s  perception  of  safety.

Consequently, the focus on bioterrorism has increased and has become a great concern.

Biological warfare is an ancient experience. Some examples in recorded history take us back to the 6th century BC. The Assyrians seemingly poisoned the wells of their opponents with rye ergot. In 1754-1763, during the French and Indian war, the English captain, Ecuyer, offered blankets smeared with smallpox to Indian loyal to the French.The exercise of biological weapons became more ubiquitous in the 20th century. During World War I the Germans tried to spread cholera and plague in Italy and St. Petersburg.

Molecular techniques to identify pathogenic organisms

There are several methods to recognize pathogenic microorganisms. These techniques are based on analyses as diverse as antigens, peptides and other ligands for identification. The key disadvantage of these methods is that they require the distinctness required to offer a positive detection. The genetic composition of all the organisms is unique. Therefore, most of the methods today stresses on the use of genetic markers for recognition and identification of bio pathogens. Any polymorphic region found in an organism’s genome which can help in positive identification is a genetic marker.

Several researchers have taken help of techniques which are based on length polymorphisms (e.g., AFLP) to effectively differentiate different bacteria species but these techniques lack the preciseness required to differentiate microorganisms which are very closely related to each other.

Q-PCR has also been used to detect and identify different pathogenic microbial and fungal species. But, the main disadvantage of PCR is its limited multiplexing capability. The method does not compete the other technologies viz. DNA microarrays or bead-based assays since they have high multiplexing ability.

Microarrays are extensively used because of their specificity, sensitivity and multiplexing capabilities. A single array chip is generally imprinted with thousands of oligonucleotides. DNA chips are used to identify and differentiate several pathogenic bacteria in the Proteus,Vibrio, Escherichia, Shigella, Salmonella, Mycobacterium and Bacillus genera.Since, microarrays require experts in bioinformatics for their design as well as analysis; it becomes very expensive for routine use. There are other problems related with the immobilization of the probes on the chip. When compared to DNA Microarrays, liquid phase assays viz. the bead-based technologies are not as vulnerable to the thermodynamic issues allied with the probe-target binding.

Liquid array analyses provide high sensitivity and high specificity, quantitative and multiplexing abilities.One of the unique bead-based liquid array systems comprises a convergence of flow cytometry and microsphere technology. In the microarray, fluorescent signals were often too feeble to analyse and had more cross-hybridization than those in the bead-based liquid array method.

Another powerful method in pathogen detection is ELISA. The difference between the Luminex assays and the ELISA is that ELISA is singleplexed and Luminexis multiplexed and more sensitive.