Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP)

Abstract

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

One-Pot Isothermal LAMP-CRISPR-Based Assay for Klebsiella pneumoniae Detection

“…Isothermal amplification is an ideal technology to overcome the disadvantages of PCR-based methods. The loop-mediated isothermal amplification (LAMP) has been widely used in pathogen detection ( 13 , 14 ). However, a relatively high rate of false-positive results is a shortcoming of the LAMP method ( 15 ).…”

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…Garg et al [ 40 ] introduced successful LAMP-based turbidimetric detection of Mycobacterium leprae targeting the 16s rRNA gene. Likewise, similar studies reported the detection of multiple pathogens—for instance, Clostridium botulinum [ 41 ], Streptococcus pneumoniae [ 42 ], and Streptococcus agalactiae [ 43 ]. However, this method provides low sensitivity, as the faint product can easily be misjudged.…”

A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens

“…However, although many long primers increase the reaction yield, a risk of primer dimerization owing to nonspecific interactions simultaneously exists [9,27]. To prevent primer dimerization, adding dimethyl sulfoxide (DMSO) and betaine is used as a strategy to decrease nonspecific interactions, thereby giving stability to oligonucleotides [28]. The use of additional compounds can amplify nucleic acids without non-specific amplification, and exponential nucleic acid amplification is achieved while forming loops within 40-60 min at a constant temperature of 60-65 • C [29].…”