Researchers have modified a device for the detection of selected foodborne pathogens in a single reaction-without any enrichment step.
Zhou et al developed and evaluated a novel application of the GenomeLab Gene Expression Profiler (GeXP) analyzer, which was capable of detecting the presence of multiple pathogens in a
single reaction.
The analytical procedure includes primer design, PCR amplification using both chimeric and universal primers, capillary electrophoretic separation based on the size of the PCR product followed by identification in terms of the expected size.
GeXP Enhancements
The GeXP multiplex PCR amplification condition was improved by using a fixed annealing temperature, including two steps with different annealing temperatures.
Step one in the first 12 cycles was performed at a higher temperature using the gene-specific sequence of the chimeric primers to produce amplicons that have universal tags.
Step two was performed using universal primers at a lower temperature in cycles 13–32. Improved PCR condition enhanced the specificity and sensitivity of multiplex PCR analysis.
Plasmid pMD19 was added to each reaction as positive control to ascertain whether the PCR step and data analysis step of the assay were carried out correctly.
Detection Speed
GeXP method was capable of detecting these six pathogens in four hours (1h for DNA extraction, 2h for PCR amplification, 45min/row of eight samples for capillary electrophoretic separation, and 10min for interpretation).
No non-specific or false-positive results were observed, according to the researchers.
The cost of the method for simultaneous identification of six foodborne pathogens is $10 per test.
GeXP-PCR assay was capable of detecting as low as 101–102 CFU/mL of six pathogens in a single tube without any enrichment steps.
For the analysis of GeXP-PCR assay, the researchers selected six of the representative bacteria associated with foodborne illnesses.
The objective was to develop a rapid, sensitive and high-throughput pathogenic bacteria diagnostic technique using GeXP-based multiplex PCR assay (GeXP-PCR) for simultaneous detection of six foodborne pathogens.
Tested Pathogens
Pathogens targeted were Salmonella enterica, Shigella spp., L. monocytogenes, C. jejuni, Staph. aureus and E. coli O157:H7.
“All six S. enterica strains, all three E. coli O157:H7 strains, four L. monocytogenes strains, four Shigella spp. strains, five Staph. aureus strains, and three C. jejuni strains were positive in the GeXP-PCR assay and all of the non-target organisms were negative in the assay.
“No mispriming or non-specific amplification was observed. None of the reactions generated more than a single peak and peaks of same species with different serotypes were shown in same position.”
The GeXP-PCR detection can combine patented XP-PCR priming strategy with capillary electrophoretic separation, resulting in high levels of specificity, sensitivity and throughput capacity.
With the GeXP, up to 35 genes can be multiplexed in a single reaction. The major advantages of this method include performance, multiplexing capability, and the ability to process samples in parallel.
Further evaluation of the GeXP-PCR assay with more food samples from different sources is necessary to confirm its value and modify the analysis procedures to reduce the number of steps involved, concluded the researchers.
Sources: Food Quality News and Food Control, Volume 32 Issue 1 July 2013 pages 198-204 (Online ahead of print, DOI: 10.1016/j.foodcont.2012.11.044).
“Simultaneous detection of six food-borne pathogens by multiplex PCR with a GeXP analyser”
Authors: Beili Zhou, Jinwen Xiao, Shengfeng Liu, Jun Yang, Yu Wang, Fuping Nie, Qing Zhou, Yingguo Li, Guohua Zhao
Abstract:
A novel application of GeXP analyzer was developed for simultaneous detection of six pathogens associated with food poisoning outbreaks, including Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, Shigella spp., and Campylobacter jejuni. Chimeric primers containing both microbe- and pMD19-specific sequences were fused to universal sequences resulted in PCR products with intended sizes. The PCR products were separated by capillary electrophoresis and identified by using fluorescence spectrophotometry. Plasmid pMD19-T was added to each reaction as positive control to ascertain the PCR steps and data analysis step of the assay. The results indicate that the GeXP method is both specific and sensitive for the detection of all six food-borne pathogens in a single reaction-without any enrichment step. In conclusion, the GeXP-PCR assay is a rapid, sensitive and high-throughput method for parallel analysis of food-borne pathogens.