Campylobacter Species Identification Using a Microchip Electrophoresis System

Significance of the topic

The accurate and rapid differentiation of Campylobacter species, particularly C. jejuni, C. coli and C. fetus, is critical in food safety and public-health laboratories for contamination source tracking and epidemiology. Multiplex PCR combined with automated fragment analysis reduces hands-on time, improves standardization of calls, and accelerates sample throughput compared with manual gel electrophoresis and visual interpretation.

Objectives and study overview

This application note demonstrates the use of the MultiNA II MCE-301 microchip electrophoresis system to analyze multiplex PCR amplicons targeting three Campylobacter species. Goals were to (1) show robust separation and size calling of species-specific amplicons and a common control fragment, (2) evaluate the system’s automated dilution to handle high-concentration PCR products, and (3) assess the Fingerprinting Analysis module for automated, objective species identification.

Methodology

Samples: DNA was extracted from bacterial isolates of C. jejuni, C. coli and C. fetus; a no-template negative control (NC) was included.

PCR assay design and targets:

• Targets and expected amplicon sizes: glyA (C. coli) 126 bp; hipO (C. jejuni) 323 bp; sapB2 (C. fetus) 435 bp; 23S rRNA (common) 650 bp.
• Primers were adopted from a published assay (Wang et al., J. Clin. Microbiol., 2002).

PCR reaction setup and cycling (summary):

• Reaction used 2x Ampdirect Plus with BIOTAQ HS DNA Polymerase to improve inhibitor tolerance; template input ~2 µL.
• Representative primer concentrations: 0.2–1.0 µM depending on primer; polymerase 0.5 U per reaction.
• Cycling: initial denaturation 95°C 10 min; 35 cycles of 94°C 30 s, 59°C 60 s, 72°C 90 s; final extension 72°C 7 min.

Electrophoresis and analysis:

• MultiNA II MCE-301 microchip electrophoresis with DNA-1000 Kit and GelStar dye.
• 100 bp DNA Ladder (pre-registered in software) used as size standard; on-chip analysis mode.
• Automated dilution function used to mitigate overloading; selectable factors 5x, 10x, 20x — a 20-fold dilution was applied to these high-concentration PCR products.
• Fingerprinting Analysis automatically compares detected fragment sizes to registered reference profiles and outputs a list of calls, while allowing simultaneous visual cross-check of electropherogram/gel image and automated calls.

Instrumentation used

• MultiNA II MCE-301 microchip electrophoresis system (Shimadzu).
• DNA-1000 Kit (P/N: 292-27911-91) with GelStar fluorescent dye (Lonza).
• 100 bp DNA Ladder (Takara Bio, P/N: 3407A) as size marker.
• PCR reagents: 2x Ampdirect Plus and BIOTAQ HS DNA Polymerase Set (Shimadzu).

Main results and discussion

Electrophoresis results demonstrated clear detection of the common 23S rRNA fragment (~650 bp) in all positive samples and distinct species-specific fragments at the expected sizes: C. coli ~126 bp, C. jejuni ~323 bp and C. fetus ~435 bp. The negative control showed no detectable amplicons, indicating absence of contamination.

The automated dilution preserved separation quality by avoiding microchip overloading, and the pre-registered ladder enabled automatic calibration for size calling. Fingerprinting Analysis produced objective species calls by matching observed fragments to reference size data; combined visual and automated review on a single screen enabled quick cross-checking and reduced subjective interpretation.

Benefits and practical applications

• Workflow automation from sample loading to data analysis reduces hands-on time and human error in routine testing.
• Automated dilution streamlines handling of high-concentration amplicons and protects the microchip, supporting instrument longevity.
• Fingerprinting Analysis standardizes interpretation and facilitates higher throughput and consistent reporting in food-safety or clinical laboratories.
• System integration supports batch processing of many samples, making it suitable for epidemiological surveillance and contamination source tracking.

Future trends and potential applications

Potential extensions and developments include:

• Higher-level multiplexing to include additional pathogens or resistance markers in a single reaction.
• Quantitative adaptations or coupling with real-time PCR to provide semi-quantitative abundance data alongside fragment sizing.
• Integration with laboratory information management systems (LIMS) for automated sample tracking and result reporting.
• Use in routine food-monitoring pipelines and outbreak investigations, with further validation for regulatory acceptance.
• Combination with sequencing or metagenomics for complex-sample analyses where culture-free identification is desired.

Conclusion

MultiNA II MCE-301 successfully analyzed multiplex PCR amplicons for three major Campylobacter species, delivering objective, automated fragment sizing and species identification. The platform’s automated dilution and Fingerprinting Analysis reduce manual steps and interpretation variability, improving throughput and standardization in laboratories performing routine microbial surveillance and food-safety testing.

References

1. Gehua Wang et al., Journal of Clinical Microbiology, 2002, 40(12), 4744–4747.