Livestock and Wildlife Laboratory (LR16IRA04), Arid Lands Institute (I.R.A), University of Gabès, Médenine 4119, Tunisia.
Laboratory of Microbiology, African Genome Centre, Mohammed VI Polytechnic University (UM6P), Lot 660-Hay Moulay Rachid, Ben Guerir 43150, Morocco.
Biosensors (Basel). 2023 Feb 20;13(2):299. doi: 10.3390/bios13020299.
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 F17-positive strains ( 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 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.
快速且特异性地检测粪便样本中的致病菌对于兽医临床新生儿腹泻的诊断至关重要。纳米抗体因其独特的识别特性，是治疗和诊断传染病的有前途的工具。在本研究中，我们报告了一种基于纳米抗体的磁荧光免疫分析方法的设计，用于灵敏检测致病性 F17 阳性菌株（F17）。为此，用纯化的 F17 菌毛 F17A 蛋白对骆驼进行免疫，并通过噬菌体展示构建了纳米抗体文库。选择了两种特异性抗 F17A 纳米抗体（Nb）来设计该生物测定。第一个（Nb1）与磁珠（MB）偶联形成复合物，能够有效地捕获靶细菌。第二个辣根过氧化物酶（HRP）缀合的纳米抗体（Nb4）用于通过氧化邻苯二胺（OPD）来检测荧光 2,3-二氨基吩嗪（DAP）。我们的结果表明，该免疫分析具有高特异性和灵敏度，在 90 分钟内检测限低至 1.8 CFU/mL。此外，我们表明该免疫分析可以应用于未经预处理的粪便样本，并且在 4°C 下储存至少一个月时仍然稳定。
