Molecular detection of toxins A, B and E of Clostridium botulinum in botulism suspected specimens

Abstract

BACKGROUND AND OBJECTIVES Botulism is caused by Clostridium botulinum toxin, which is produced by improper use of canned goods, dairy products, and fish.Clostridium botulinum toxin (BONT) has seven designated serotypes (A-B-C-D-E-F-G) that have a similar structure but differ antigenically. Serotypes A, B, and E and rarely F are associated with human clinical cases. Today, in Iran, a definitive diagnosis of botulism is made by identifying the bacterial toxin using the laboratory method of injection into laboratory mice (mouse bioassay). The rapid onset of botulism toxin poisoning and the resulting clinical symptoms show the need for quick and accurate disease diagnosis techniques and methods that play an effective role in saving the lives of those affected. In addition to being fast, accurate, and economical, these diagnostic methods must have high specificity and sensitivity. The purpose of this research is to use the Real-time PCR molecular method to detect Clostridium botulinum bacteria in a clinical sample. MATERIALS AND METHODS In 6 months, 50 clinical samples (feces, patient's stomach secretions, and food samples) suspected of botulism, which are sent from all over the country to the botulism laboratory of Pasteur Institute of Iran, were collected. After 48 hours of cultivation in Cook meat environment and under anaerobic conditions and examination of slides, DNA extraction was done with a kit and finally SYBR Green Real-Time test was done for the genes of toxins A, B, and E and the results of that case Analysis was done. RESULTS AND DISCUSSION In this research, out of the 50 samples that were studied, 68% were molecularly positive and mouse bioassay negative, 12% were positive in both, and 10% of the samples were molecularly positive but negative in the slide. The frequency of toxins B, A, BE, E, and AB of Clostridium botulinum was 44%, 32%, 22%, 16%, and 10%, respectively. The difference between the results of the SYBR Green Real-Time test compared to the Mouse bioassay test can be due to the high sensitivity of the molecular test to the phenotype as well as the lack of expression of toxin genes in some samples. CONCLUSION Due to the high sensitivity and accuracy of the SYBR Green Real-Time method and the short time of this test, it is recommended to use this method along with the Mouse bioassay method to detect botulism disease toxin