PATHOGEN DETECTION
Identification of The Microscopic Killers
Methods of Detection
Polymerase Chain Reaction (PCR)
One of the most common molecular based methods of detection is PCR. Invented about 30 years ago, it detects a specific DNA sequence of an infectious bacteria present. It works by strengthening the targeted DNA sequence in a three step cycle. First, the DNA is denatured (altered with heat) from double stranded to single. Then, the two strands have primers attach to the complementary portions of the original. The primers also attach the loose
loose nucleotides to their complementary strands to copy the DNA. This process has used in the detection of E. coli and Salmonella. To detect the PCR products it needs to be run with an agar gel electrophoresis.
Multiplex PCR (mPCR)
Multiplex PCR is like regular PCR, but it is a faster detection because it strengthens multiple target genes. The basic process is similar to simple PCR, however there are multiple sets of specific primers while simple PCR only uses one specific set.
Before mPCR was only able to detect two to three pathogens at a time but it has advanced to detect five or more pathogens simultaneously. Currently there is progress in developing technology that can detect more pathogens.
Quantitative PCR (qPCR)
Real-time or quantitative PCR is different than simple PCR in that there is no need to run the products in agarose gel electrophoresis to detect the products. It has the ability to track the entire process of forming PCR products by measuring the fluorescent signals made by probes and dyes. The intensity is relative to the amount of DNA.
Due to not having the post-PCR process of running it through a gel, cross contamination was lowered and there is less time used up from the labor. Because of this there have been commercial products of qPCR.
Nucleic acid sequence-based amplification (NASBA)
NASBA works by strengthening the nucleic acids under a constant temperature unlike PCR where the temperature is constantly changing. Usually this process is used in strengthening
the concentration of RNA, which will be used as a
template to copy DNA. The reaction happens at around 41°C with three enzymes and two specific primers. The products, however needed to be detected with gel electrophoresis.
That extra step led to the development of real-time NASBA which used fluorescent probes like qPCR had to constantly examine it. Real-time NASBA also allowed for detection of surviving pathogens.
Loop-mediated isothermal amplification (LAMP)
The LAMP process is fast, specific, and sensitive to the type of pathogen it is detecting. It is an auto cycle and runs its DNA synthesis under constant temperatures of 59°C and 65°C for 60 min. LAMP uses four primers; two inner and two outer to target six specific DNA positions. A large amount of DNA or RNA is able to be made within a short amount of time. The LAMP products still though need to be detected with gel electrophoresis. It was proven that LAMP is more sensitive than PCR in detecting food borne pathogens.
Optical Biosensors
Surface plasmon resonance (SPR) is the most common form of optical biosensors. SPR works by utilizing spectroscopy for pathogen detection. The bioreceptors are placed on the surface of a thin metal sheet. Electromagnetic radiation interacts with the electron cloud of the metal, but when a pathogen is put on it it changes the wavelength of the electron resonance.
Commercial use of SPR is detecting E. coli on ground beef, milk, and apple juice. It is also used in the detection of Salmonella.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is one of the most common methods that is immunological based for detecting pathogens. Sandwich ELISA is the most effective type of ELISA by involving only two types of antibodies. The main antibody is disabled on the walls of the microtiter plate wells. The targeted antigens of the pathogen bind themselves to the antibodies, while the rest of the unbound antigens are taken out.
Afterwards, another antibody is added that will bond with the antigen attached to the first antibody. The excess antigens are taken out, leaving only the "squished" antigens. These "squished" antigens can be detected with the use of a colorless substrate that will change color in the presence of the enzyme. The use of ELISA is a rapid form of detection and is now used commercially.
Lateral Flow Immunoassay
Lateral flow immunoassay were developed to be rapid, cheap, and simple yet reliable results due to ELISA method needing specialized equipment and trained personnel. An example of this method is a dipstick and immunochromatographic strips that allow for fast on site detection of the pathogen.
A device is made of four sections arranged on a plastic backing, sample pad at the bottom, conjugate pad, a nitrocellulose sheet and finally an absorbent pad. The sample will move through with capillary action and reacts with the conjugate.