Qiuying Huang1, Qinghua Hu, Qingge Li. 1. Molecular Diagnostics Laboratory, Department of Biomedical Sciences, Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
Abstract
BACKGROUND: Real-time PCR assays have been widely used for detecting foodborne pathogens but have been much less frequently applied in species identification, mainly because of the low number of species they can distinguish in 1 reaction. The present study used a new probe coding/labeling strategy, termed multicolor combinational probe coding (MCPC), to increase the number of targets that can be distinguished in a single real-time PCR for rapid and reliable species identification. METHODS: With MCPC, 8 pairs of species-specific tagged primers, 1 pair of universal primers, and 8 unilabeled or mix-labeled molecular beacon probes were included in a single reaction tube. Real-time PCR was performed, and the identity of each of the 8 pathogens was determined by amplification profile comparison. The method was validated via blind assessment of 118 bacterial strains, including clinical isolates and isolates from food products. RESULTS: The blind test with 118 samples gave no false-positive or -negative results for the target genes. The template DNA suitable for MCPC analysis was simply prepared by heating lysis, and the total PCR analysis was finished within 2.5 h, excluding template preparation. CONCLUSIONS: MCPC is suitable for rapid and reliable identification of foodborne pathogens at the species level.
BACKGROUND: Real-time PCR assays have been widely used for detecting foodborne pathogens but have been much less frequently applied in species identification, mainly because of the low number of species they can distinguish in 1 reaction. The present study used a new probe coding/labeling strategy, termed multicolor combinational probe coding (MCPC), to increase the number of targets that can be distinguished in a single real-time PCR for rapid and reliable species identification. METHODS: With MCPC, 8 pairs of species-specific tagged primers, 1 pair of universal primers, and 8 unilabeled or mix-labeled molecular beacon probes were included in a single reaction tube. Real-time PCR was performed, and the identity of each of the 8 pathogens was determined by amplification profile comparison. The method was validated via blind assessment of 118 bacterial strains, including clinical isolates and isolates from food products. RESULTS: The blind test with 118 samples gave no false-positive or -negative results for the target genes. The template DNA suitable for MCPC analysis was simply prepared by heating lysis, and the total PCR analysis was finished within 2.5 h, excluding template preparation. CONCLUSIONS: MCPC is suitable for rapid and reliable identification of foodborne pathogens at the species level.