Multiplex quantitative foodborne pathogen detection using high resolution CE‐SSCP coupled stuffer‐free multiplex ligation‐dependent probe amplification

Abstract

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection.

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Long stuffer sequences affect the quantitative consistency because the amplification efficiency fluctuates between short and long products. Thus, Chung et al [65] developed stuffer-free MLPA-CE-SSCP to sensitively detect 10 foodborne pathogens. All 10 pathogens were reliably identified, with LODs of 0.5-5 pg of genomic DNA.…”

Accurate and effective multidrug-resistant Mycobacterium tuberculosis detection method using gap-filling ligation coupled with high-resolution capillary electrophoresis-based single strand conformation polymorphism

“…To detect multiple GFL products, CE-SSCP with highly enhanced resolution and separation was adopted. GFL products could be separated according to their unique folding structures depending on the sequence variations in the capillary filled with poly (ethyleneoxide)–poly (propyleneoxide)–poly (ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, which we previously reported262728. To establish this MDR/XDR-TB detection system, we selected highly polymorphic regions as targets for multiplex detection (Table 1).…”

Clinical application of a ligation-independent pathway of multiplex ligation-dependent probe amplification for the determination of quinolone susceptibility of Streptococcus pneumoniae

“…Several studies have shown that MLPA can be applied to identify and characterize multiple microorganisms (Bergval et al, 2008, Chung et al, 2012, Terefework et al, 2008, Wolffs et al, 2009. Although MLPA has been used in many clinical applications, its disadvantages limit the general use of this technique in clinical laboratories.…”