Table 1.

Methods for foodborne pathogen detection.

Method	Target Groups	Principle	Advantages	Limitations
Culture Based Methods	Bacteria, fungi, and viruses	Use of culture media containing the growth requirements of target microorganisms	High success rate for culturable isolates Reliable and cost-effective Can be used to target specific microbial groups & differentiate different groups Microbial cultures can be used for other applications or downstream processes	Misses large amounts of microbial groups that cannot be cultured (low sensitivity Slow-growing cultures are disadvantaged Slow turnover and laborious processes Not suitable for the rapid detection of microorganisms
Immunological assays (ELISA and LFD)	Bacteria, fungi, and viruses	The affinity between microbial antigens and monoclonal or polyclonal antibodies is exploited for the detection of microorganisms	Easy to carry out as the process can be automated improving efficiency and making it less labour intensive Large number of samples can be processed at once Can be highly specific, Toxins can be detected	False positives can be generated Limited microbial coverage Require the use of trained personnel antibodies/antigens must be labelled
Conventional PCR (e.g., nested, touchdown, multiplex, etc.)	Bacteria, fungi, and viruses	Based on the use of primers targeting specific regions of microbial DNA. Target when present is exponentially amplified	Highly sensitive and specific Reliable and widely used Can be automated Can be used to detect multiple microbial groups in a single reaction	Primer design is very important Difficult to use to distinguish between viable and non-viable cells Sensitive to contamination and this may lead to false positives Sensitive to inhibitors which may lead to false negatives
Viability PCR/qPCR	Bacteria	Special dyes used in the test render non-viable bacterial DNA non-amplifiable	Dead and viable bacterial cells can be easily differentiated Viable cells can be quantified Faster than culture-based approaches	False positives can result Use of mRNA more reliable than DNA Not as widely used as other methods
Reverse Transcriptase (RT) PCR/qPCR	Bacteria, fungi, and viruses	mRNA transcripts level declines quickly after cell death. Detected mRNA should be from viable cells and is therefore targeted (RT-PCR). Transcripts are amplified and quantified using dyes or probes	Relatively quicker than culture-based approaches Reliable and widely used Highly sensitive and specific Quantification of cells (absolute number or gene copy numbers) can be achieved	False positives can occur as not all mRNAs are short-lived Requires the use of trained personnel Can be expensive to run Not suited for use when samples yield < 200 bp products Sensitive to contaminants and inhibitors
Next Generation Sequencing Approaches	Bacteria, fungi, and viruses	Whole genome or markers such as ITS and 16S rRNA are targeted and sequenced (random shotgun sequencing, gene/marker specific sequencing, etc.)	Reliable and accurate results and also now more widely used Suitable for detection/identification of all groups of microorganisms (rare, fastidious & unculturable groups Predictable turn-around times Large number of samples can be analyzed Generates a huge amount of useful data Continuous improvement of technologies and platforms	Expensive to set up and run compared to other methods Requires trained personnel to run and analyze the results Result are as good as the reference database

Adapted from [3,6,15,16,17,37,38,39,40].