Rapid, Sensitive, and Specific Detection of Clostridium tetani by Loop-Mediated Isothermal Amplification Assay

Abstract

Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

Rapid Detection of Clostridium tetani by Recombinase Polymerase Amplification Using an Exo Probe

“…Exo-RPA is a highly efficient amplification method and exhibits great potential to minimize the detection turnaround time. LAMP, introduced by Jiang [ 21 ], was analyzed through turbidimetry and electrophoresis on agarose gels with analytical sensitivities ranging from 10-10 6 CFU/ml. According to E. Cohen [ 37 ], the gene encoding the neurotoxin was not identified in ATCC 19406.…”

“…The abovementioned methods present a few disadvantages, including relatively low sensitivity and specificity, and cannot provide accurate quantification. Various quantitative approaches have been established, like real-time polymerase chain reaction (PCR) [ 20 ] and loop-mediated isothermal amplification assays (LAMP) [ 21 ], to determine the fragments of the TeNT gene. PCR-based assays require automated thermocycling equipment and trained personnel, which limit their potential clinical applications in resource-poor regions.…”

A simple and sensitive electrochemical sensor for rapid detection of Clostridium tetani based on multi-walled carbon nanotubes

“…The oligonucleotides used in this work to design primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), and their sequences are listed in Table 1. 2 MWCNTs were purchased from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Sciences (Chengdu, China). HAuCl 4 , b-nicotinamide adenine dinucleotide hydrate (NADH), bovine serum albumin (BSA), and hexanethiol (HT) were obtained from Sigma-Aldrich Co., LLC (Shanghai, China).…”

“…In patients afflicted with war injuries, the contamination rates of tetanus vary between 25% and 80%. 2 Seale A. C. et al estimated that the mortality rate of neonatal tetanus was approximately 62.2% in South Asia, sub-Saharan Africa, and Latin America in 2010. 3 C. tetani is a Gram-positive, anaerobic, rod-shaped bacterium that can develop a terminal spore.…”

“…In this work, the iRCA assay used fragment C of the tetanus neurotoxin gene as the target gene. 2 Undoubtedly, this electrochemical biosensor greatly broadens the application of the iRCA-based sensing technique and can be utilized to detect various target molecules via the process of iRCA, a sensing mechanism for C. tetani based on the iRCA technique. It can further reduce the time requirement of the conventional RCA method and improve the sensitivity and specicity.…”