Detection of Soybean-Derived Components in Dairy Products Using Proofreading Enzyme-Mediated Probe Cleavage Coupled with Ladder-Shape Melting Temperature Isothermal Amplification (Proofman–LMTIA)

Xiao, Fugang; Gu, Menglin; Zhang, Yaoxuan; Xian, Yaodong; Zheng, Yaotian; Zhang, Yongqing; Sun, Jian; Ding, Chen; Zhang, Guozhi; Wang, Deguo

doi:10.3390/molecules28041685

Cited by 10 publications

(2 citation statements)

References 30 publications

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“…LMTIA product detection relies on increasing fluorescence during amplification via the Proofman probe(s), enhancing accuracy through sequence-specific binding and cleavage [12]. This Proofman-LMTIA technique has been successfully applied in the rapid detection of harmful bacteria Listeria monocytogenes contamination in food [12] and soy-derived components in dairy products [13].…”

Section: Introductionmentioning

confidence: 99%

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique

Zhang,

Li,

Zhang

et al. 2023

Molecules

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This study aims to establish a rapid identification method based on the Proofman-LMTIA technique for distinguishing between Panax quinquefolium and Panax ginseng. By targeting specific 18S rDNA sequences, suitable primers and Proofman probes labeled FAM or JOE were designed for LMTIA. Initially, single-species-primer Proofman-LMTIA assays were performed separately for each ginseng type to optimize reaction temperature, assess sensitivity and specificity, and determine the detection limit. Subsequently, both sets of primers and their corresponding probes were combined in the same reaction system to further optimize reaction conditions, evaluate sensitivity, and assess stability. Finally, the developed Proofman-duplex-LMTIA technique was employed to detect P. quinquefolium and P. ginseng slices available in the market. Single-plex Proofman-LMTIA assays revealed that the optimal reaction temperature for both P. quinquefolium and P. ginseng was 62 °C. The sensitivity was as low as 1 pg/μL, with a detection limit of 0.1%, and both showed excellent specificity. The optimal temperature for Proofman-duplex-LMTIA assays was 58 °C. This method could simultaneously identify P. quinquefolium and P. ginseng. Testing 6 samples of P. ginseng and 11 samples of P. quinquefolium from the market resulted in a 100% positive rate for all samples. This study successfully established a rapid, simple, sensitive, and specific Proofman-duplex-LMTIA identification method for P. quinquefolium and P. ginseng. It provides an effective means for quality control of P. quinquefolium, P. ginseng, and related products.

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Section: Introductionmentioning

confidence: 99%

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique

Zhang,

Li,

Zhang

et al. 2023

Molecules

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“…In this study, we aim to develop a rapid, sensitive, and specific detection method for Listeria monocytogenes by combining a novel probe, Proofman probe (proofreading enzyme-mediated probe cleavage) [ 23 , 24 , 25 ], with LMTIA technology. When using a Proofman probe, an additional DNA polymerase (Pfu) with proofreading activity was involved to cleave the probe from the 3′ end, producing a fluorescent signal.…”

Section: Introductionmentioning

confidence: 99%

Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay

Song

Wang

et al. 2023

Molecules

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Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers’ specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method’s applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.

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