A 4-plex Droplet Digital PCR Method for Simultaneous Quantification and Differentiation of Pathogenic and
Shuwen Lei1,2, Xiaokui Gu3,4, Wei Xue3
1Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, China.
Abstract:
Vibrio parahaemolyticus is a significant seafood-borne pathogen, leading to serious acute gastrointestinal diseases worldwide. In this study, a reliable 4-plex droplet digital PCR (ddPCR) was successfully established and evaluated for the simultaneous detection of V. parahaemolyticus based on tlh, tdh, ureR, and orf8 in food samples using single intact cells. The targets tlh and ureR were labeled with 6-Carboxyfluorescein (FAM), and the targets tdh and orf8 were labeled with 5'-Hexachlorofluorescein (HEX). Due to reasonable proration of primers and probes corresponding into the two fluorescence channels of the ddPCR detecting platforms, the clearly separated 16 (24) clusters based on fluorescence amplitude were obtained. For better results, the sample hot lysis time and the cycle number were optimized. The results showed that the minimum number of "rain" and maximum fluorescence amplification were presented for precise detection in the condition of 25 min of the sample hot lysis time and 55 cycles. The sensitivity of this 4-plex ddPCR assay was 39 CFU/mL, which was in accordance with that of the conventional plate counting and was 10-fold sensitive than that of qPCR. In conclusion, the 4-plex ddPCR assay presented in this paper was a rapid, specific, sensitive, and accurate tool for the detection of V. parahaemolyticus including pandemic group strains and could be applied in the differentiation of V. parahaemolyticus in a wide variety of samples.
Insights
A new 4-plex droplet digital PCR (ddPCR) assay accurately detects Vibrio parahaemolyticus, a common seafood pathogen. This method offers enhanced sensitivity and specificity for identifying bacteria in food samples.
Area of Science:
- Food Microbiology
- Molecular Diagnostics
- Pathogen Detection
Background:
- Vibrio parahaemolyticus is a major cause of seafood-borne gastrointestinal illness globally.
- Accurate and sensitive detection methods are crucial for food safety and public health.
- Existing methods may lack the sensitivity or specificity required for comprehensive detection.
Purpose of the Study:
- To develop and validate a reliable 4-plex droplet digital PCR (ddPCR) assay.
- To enable simultaneous detection of Vibrio parahaemolyticus using specific target genes (tlh, tdh, ureR, orf8).
- To assess the assay's performance in food samples at the single-cell level.
Main Methods:
- Established a 4-plex ddPCR assay targeting four Vibrio parahaemolyticus genes: tlh, tdh, ureR, and orf8.
- Utilized dual-color fluorescence labeling (FAM and HEX) for multiplex detection.
- Optimized sample hot lysis time (25 min) and cycle number (55) for precise results.
Main Results:
- Achieved clearly separated clusters (16 distinct populations) based on fluorescence amplitude.
- Demonstrated a sensitivity of 39 CFU/mL, comparable to plate counting and 10-fold higher than qPCR.
- Confirmed the assay's ability to detect Vibrio parahaemolyticus, including pandemic strains, in food matrices.
Conclusions:
- The developed 4-plex ddPCR assay is a rapid, specific, sensitive, and accurate tool for Vibrio parahaemolyticus detection.
- The assay is suitable for differentiating Vibrio parahaemolyticus in diverse food samples.
- This method provides a significant advancement in molecular diagnostics for foodborne pathogens.
