[en] Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.
Disciplines :
Microbiology
Author, co-author :
Dhehibi, Asma; Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Allaoui, Abdelmounaaim; Laboratory of Microbiology, African Genome Centre, Mohammed VI Polytechnic University (UM6P), Lot 660-Hay Moulay Rachid, Ben Guerir 43150, Morocco
Raouafi, Amal; Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), University of Tunis El Manar, Tunis El Manar 2092, Tunisia
Terrak, Mohammed ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP)
Bouhaouala-Zahar, Balkiss ; Laboratory of Venoms and Theranostic Applications (LR20IPT01), Place Pasteur, BP74, Pasteur Institute of Tunis, University of Tunis El Manar, Tunis 1002, Tunisia
Hammadi, Mohamed; Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Raouafi, Noureddine ; Sensors and Biosensors Group, Analytical Chemistry and Electrochemistry Lab (LR99ES15), University of Tunis El Manar, Tunis El Manar 2092, Tunisia
Salhi, Imed ; Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia
Language :
English
Title :
Nanobody-Based Sandwich Immunoassay for Pathogenic Escherichia coli F17 Strain Detection.
MHESR - Ministry of Higher Education and Scientific Research
Funding text :
This work was supported by PRF program of The Tunisian Ministry of Higher Education and Scientific Research (NanofastResponse, grant ref.: PRF2017D4P1).
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