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

Abstract

Previous research indicates that the structural gene coding for the neurotoxin is on a plasmid [1,2]. The neurotoxin can be endocytosed by both Rab5-and clathrin-dependent mechanisms and then transported to the central nervous system (CNS) by long-distance retrograde axonal transport, where it can inhibit the release of neurotransmitters in the spinal cord, leading to periodic hyper contraction of skeletal muscles (termed spastic paralysis) [3]. The spores of C. tetani are abundantly found in soil and other environments; in most cases, tetanus originates from damaged skin or narrow wounds, such as those resulting from dental infections, intravenous drug abuse, compound fractures, or nail punctures. These areas confer a more favorable anaerobic environment for the growth and reproduction of C. tetani [4,5]. Tetanus can be prevented by active immunization of children and pregnant women. There is no effective treatment for this disease. Tetanus management involves aggressive airway management, treatments to inhibit the absorption of toxins, strict sanitary measures, treatments to alleviate muscle spasms, high-intensity therapy, and intensive supportive care [6,7]. Tetanus generally occurs in low and middle-income countries but has a low incidence in developed countries [6,8]. Maternal and neonatal tetanus (MNT) maintains a widespread public health issue, with mortality between 80% and 100% among neonates [9]. It is estimated that one million cases of tetanus happen worldwide annually and led to between 48199 and 80042 deaths in 2015. The World Health Organization (WHO) estimated that 30,848 newborns died of neonatal tetanus in 2017, and the imputed number of neonatal tetanus deaths decreased to 25000 in 2018 [10,11]. Although mortality is steadily declining, we believe that the global incidence of the disease is underestimated because most cases arise in resource-limited settings where surveillance systems are limited, and accurate data cannot be obtained. Over the past few decades, intensive care measures have undergone significant changes in numerous countries, and it is possible that high tetanus incidence rates persist despite the remarkable declines in mortality that have occurred [12][13][14]. This is an additional factor that may result in an underestimation of disease.The diagnosis of tetanus typically relies on the clinical history and major symptoms. In localized tetanus, differential diagnosis is necessary, and laboratory tests can exclude any other diseases with similarities to tetanus, such as strychnine poisoning, neuroleptic malignant syndrome, stiff-person syndrome, and dystonic reaction to Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to ...

Clostridium tetanibacteraemia in the plague area in France: two cases

“…Isolated C. tetani from Ind-7 and Ind-44 were deposited in the Collection de Souches de l’Unité des Rickettsies (IHU Méditerranée Infection, Marseille, France) under reference numbers CSUR Q7451 and CSUR Q7452, respectively. Further, toxigenic C. tetani strains were confirmed by aDNA PCR amplification and sequencing of a 240-bp TeNT fragment in Ind-7 and Ind-44 dental pulps (data not shown)(11). C. tetani was not detected by the two different paleoculturomics and paleomicrobiology approaches in the sediments and calculus surrounding the C. tetani -positive teeth.…”

“…To confirm the presence of C. tetani in the ancient pulps, a PCR targeting a 240-bp tetanospasmin (tent) gene of C. tetani was performed, using the following primers set by Guo et al , 2012 (11): F1-ATGCGCCATCGTATACTAAC; R1-CCATCTTTCGGATAACCTACA; F2-TATGTATTTGACAAATGCG; R2-CTTTCGGATAACCTACAAT. The thermal profile included an initial five-minute denaturation step at 95 °C followed by 45 cycles consisting of 30 seconds of dissociation at 95 °C, 30 seconds of annealing at 51 °C and 60 seconds elongation at 72 °C.…”

Agarose Hydrogel-Boosted One-Tube RPA-CRISPR/Cas12a Assay for Robust Point-of-Care Detection of Zoonotic Nematode Anisakis

“…Recently, the DETECTR method has been established by combining the CRISPR/Cas system with the recombinase polymerase amplification (RPA) system for highly sensitive and specific nucleic acid detection. The RPA system can amplify targeted double-stranded DNA (dsDNA) isothermally at 37–42 °C without a thermocycler, showing great suitability for on-site testing . The RPA assay could improve the sensitivity of the CRISPR/Cas12a assay .…”

“…The RPA system can amplify targeted double-stranded DNA (dsDNA) isothermally at 37−42 °C without a thermocycler, showing great suitability for on-site testing. 12 The RPA assay could improve the sensitivity of the CRISPR/Cas12a assay. 13 On the other hand, the CRISPR/Cas12a system could boost the specificity of RPA, and the sequence-specific recognition of the CRISPR system could reduce false-positive results caused by the RPA products.…”

Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification

“…Although this agarose gel electrophoresis combined with RPA technique has been used in some pathogens detection, it is not optimistic for POCT detection. In addition, more and more literatures have combined RPA with lateral flow test strip (LFS) or fluorescence method to achieve real-time visualization of results ( Ji et al., 2023 ; Guo et al., 2022 ). Although some portable fluorescence reading devices have been widely used, considering the cost of the instrument, this study did not adopt the experimental design combined with fluorescence method at the beginning of design.…”