RESUMEN
Background & objectives: Botulism, a potentially fatal paralytic illness, is caused by the botulinum neurotoxins (BoNTs) secreted by Clostridium botulinum. It is an obligate anaerobic, Gram-positive, spore-forming bacterium. BoNTs are classified into seven serotypes based on the serological properties. Among these seven serotypes, A, B, E and, rarely, F are responsible for human botulism. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA)-based detection system for the detection of BoNT/E. Methods: The synthetic gene coding the light chain of BoNT serotype E (BoNT/E LC) was constructed using the polymerase chain reaction primer overlapping method, cloned into pQE30UA vector and then transformed into Escherichia coli M15 host cells. Recombinant protein expression was optimized using different concentrations of isopropyl-?-D-1-thiogalactopyranoside (IPTG), different temperature and the rBoNT/E LC protein was purified in native conditions using affinity column chromatography. The purified recombinant protein was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by western blot and matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF). Polyclonal antibodies were generated against rBoNT/E LC using Freund's adjuvant in BALB/c mice and rabbit. Sandwich ELISA was optimized for the detection of rBoNT/E LC and native crude BoNT/E, and food matrix interference was tested. The developed antibodies were further evaluated for their specificity/cross-reactivity with BoNT serotypes and other bacterial toxins. Results: BoNT/E LC was successfully cloned, and the maximum expression was achieved in 16 h of post-induction using 0.5 mM IPTG concentration at 25癈. Polyclonal antibodies were generated in BALB/c mice and rabbit and the antibody titre was raised up to 128,000 after the 2nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5�[3] MLD50 of native crude BoNT/E at a dilution of 1:3000 of mouse (capturing) and rabbit (revealing) antibodies. Further, different liquid, semisolid and solid food matrices were tested, and rBoNT/E LC was detected in almost all food samples, but different levels of interference were detected in different food matrices. Interpretation & conclusions: There is no immune detection system available commercially in India to detect botulism. The developed system might be useful for the detection of botulinum toxin in food and clinical samples. Further work is in progress.
RESUMEN
Background & objectives: Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler. Methods: We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included. Results: The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 μl reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 μl reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods. Interpretation & conclusion: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.