Table 2.

Comparative analysis of the nucleic acid amplification-based methods for detecting foodborne pathogens in food samples.

Amplification method	Advantages	Disadvantages	Possible improvement
Conventional PCR	-High sensitivity and specificity -Faster than cultured based method -Able to multiplexing -Able to distinguish between the bacterial strains within the same species -Commercial kits are available abundantly -Primer design software is available offline and accessible online	-Requires thermo-cycling machine -Time-consuming as it may still require enrichment step and DNA purification -Manual operations and cumbersome peripheral equipment -Amplicon can only be analyzed after PCR process -Highly dependent on the quality of samples containing nucleic acids for amplification -Prone to PCR inhibitors -Cross contamination may occur -Lack of quantitative capacity -Optimization for multiplexing is difficult and can lead to increased cost	-Automated instrument replacing manual operations from sample preparation to detection -Design of reagents that increase the PCR’s specificity and multiplexing capability as well as reduce cross-contamination, less sensitive to PCR inhibitor -Integration with microfluidic platforms or other advanced technologies
qPCR	-High sensitivity and specificity -Faster than cultured based and conventional PCR method -Does not require post-amplification process -Allow real-time monitoring for amplification -Enable the detection and quantification simultaneously of target pathogens -High throughput due to software driven operation	-Requires thermo-cycling machine -Highly dependent on the quality of samples containing nucleic acids for amplification -High cost for instrument, reagent, fluorescent dye or fluorescent probe -Prone to PCR inhibitors -Necessity for creating standard curve -Emission spectra overlapping and nonspecific binding -Optimization for multiplexing is difficult and can lead to increased cost -Required trained personnel	-Automated instrument replacing manual operations from sample preparation to detection -Design of reagents that increase the PCR’s specificity and multiplexing capability as well as reduce cross-contamination, less sensitive to PCR inhibitor -Design of affordable and user-friendly instrument
Digital PCR	-Highly precise and accurate quantification of target molecules in complex samples -More accurate and reproducible than qPCR -Enable the detection and quantification simultaneously of target pathogens -High throughput due to software driven operation -Able to distinguish the DNA from dead and viable cells by using DNA-binding fluorescent dyes	-Requires thermo-cycling machine -High cost of instrumentation -Highly dependent on the quality of samples containing nucleic acids for amplification -Requirement for thermal cycler -Prone to PCR inhibitors -False positive results due to the presence of non-target DNA or RNA -Requires analytical droplets or chambers for the application of Poisson distribution -Complexity in designing appropriate dilution of the sample to generate accurate data -Limited multiplexing capabilities compared to qPCR -Require technical expertise	-Optimization and standardization of reaction conditions, sample preparation, and data analysis -Optimization of multiplexing capacity -Improvement of partitioning using microfluidic devices
LAMP	-Suitable for amplifying and detecting larger nucleic acids -High sensitivity and specificity -Rapid detection (less than 1 h) -More sensitive or at least comparable to conventional PCR or qPCR -High amplification efficiency -Does not require an expensive thermocycling instrument -Inexpensive instrument of amplification -Resistant to LAMP inhibitors -Support for multiplexing -Rapid and simple procedure compared with PCR methods -Cheaper than PCR-based methods -Amplification results can be accessed using fluorescent intercalating dyes or colorimetric measurement	-Requires multiple primers -primers interaction could lead to false-positive results -Formation of non-specific amplification and primer dimerization -Visual inspection or turbidity measurement of the LAMP product could lead to misjudgment -Aerosol pollution during LAMP reaction -Maintenance of the stability of LAMP reagents and primers -Tedious sample preparation	-Optimization of reaction conditions -The use of artificial intelligence (AI) for evaluating the color different in visual detection -Automated instrument replacing manual operations by integrating with microfluidic devices to reduce cross-contamination
RPA/RAA	-Rapid detection (less than 30 min) -Low-temperature requirement for amplification -The sensitivity and specificity comparable to conventional PCR/qPCR -Inexpensive instrument -Support for multiplexing -Capability of amplification without the need for DNA extraction or purification -Stable reagents at room temperature -Potential use for on-site testing -Tolerant to common amplification inhibitors -Capable of amplifying target nucleic acid from a minimal processing sample	-Requires multiple enzymes -High cost for the recombinase-primer complex -Requirement for technical expertise -Prone to non-specific amplification -Poor resolution for quantification -Affected by high DNA concentration -Stringent reaction conditions -Tedious sample preparation	-Development of the next generation of RPA/RAA technology with improved sensitivity and specificity
RCA	-High throughput detection -The sensitivity and specificity comparable to conventional PCR/qPCR -Support for multiplexing	-Highly dependent on the quality of samples containing nucleic acids for amplification -The synthesis cost is relatively high -Susceptible to background signal interference	-Design of affordable reagents that increase the RCA’s specificity and multiplexing capability
SRCA	-High specificity and high sensitivity -Sample enrichment can enhance detection sensitivity and has the potential to exceed that of conventional PCR or qPCR -Does not require padlock probe and ligase -Amplification results can be assessed visually by the presence of white precipitate or by fluorescence measurement -Simpler and cheaper than LAMP and SPIA	-Limited commercial kits for DNA purification and SRCA reactions -The necessity to select an appropriate primer pair from a substantial pool of designed primers that have been extensively validated through experimentation	-Design of affordable reagents that increase the RCA’s specificity and multiplexing capability
NASBA	-Sensitive and specific -No requirement for denaturation steps -Can directly amplify RNA fragments -DNA residues in samples will not yield a false positive signal -Potential method for the quantification of viable bacteria	-Difficulties in handling RNA -Require complex equipment for the detection of the amplified RNA products -Some food substrates inhibit the reaction -Limited by RNA secondary structure -Limited length of target sequence -Requires multiple enzymes -Enzymes are not thermostable -Less efficient in amplifying longer RNA targets -Requires a precise temperature control	-User friendly design for the amplicon analysis -User friendly application for primer design -Design of affordable reagents that increase the NASBA’s multiplexing capability
SDA	-Good sensitivity and specificity -Low equipment requirements -Simple and easy-to-control workflow	-Requires for an additional thermal denaturation for analyzing DNA -Difficult to amplify long fragment -Prone to contamination from enzymes	-Simplification of SDA procedures -Development of effective methods to reduce the contamination from the enzymes -Integration with CRISPR/Cas-based technology to improve the sensitivity and specificity detection
EXPAR	-Sensitive and specific -Combination with immunomagnetic beads and aptamer can improve the sensitivity and specificity -Rapid amplification -Does not require DNA extraction -Inexpensive instrumentation	-Complexity in designing a standard EXPAR template -Nonspecific interactions of EXPAR templates with interference DNA sequences in the sample matrix may trigger background amplification -Use high concentrations of DNA template -Not suitable for the amplification and detection of long nucleic acids	-Development of effective methods to reduce or eliminate background amplification -Simplification of EXPAR procedures
SPIA	-High specificity and high sensitivity -Effectively avoid contamination of amplified products -The amplification products can be monitored using a real-time amplification fluorescence curve or assessed through visible fluorescence observed under natural daylight without the need for specialized equipment	-High cost for obtaining the required enzymes -Complicated experimental procedure could lead to a longer total assay time	-Simplification of SPIA procedures -Design of
HDA	-Good sensitivity -Suitable for amplifying and detecting larger nucleic acids -Ability for analyzing long DNA targets -Supports for multiplexing -Simple primer design, easy operation	-Non-suitability for analyzing samples with less than 100 copies -Involves multiple enzymatic steps and temperature cycling -Detection is less sensitive than PCR-based methods -Commercial HDA kits are limited	-Design of affordable reagents that increase the HDA’s multiplexing capability
CPA	-High sensitivity -Easy operation -Low equipment requirements -Does not require a DNA denaturation step -Has great potential for on-site, field and in-situ assay applications	-Requires 5 primers -Complexity in primer design -Complexity of reaction components -Difficulties in visualization the results and the complexity of result analysis	-Integration with a simple readout method, such as lateral flow assay -Integration with other technologies, such as biosensors, to simplify the PCA procedures

IMS, immunomagnetic separation; CE, capillary electrophoresis; MCE, microchip electrophoresis, PCR, polymerase chain reaction; CRISPR, clustered regularly interspaced short palindromic repeats; ELISA, enzyme-linked immunosorbent assay; ELGA, enzyme-linked gel assay; ECL, electrochemiluminescent.