Detection Of Fusobacterium Nucleatum Using Magnetic Nanoparticle Probe-based Dark-field Count Method

Fusobacterium nucleatum（F.nucleatum）,an opportunistic pathogen that mainly inhabits in the oral cavity and digestive tract of humans or various mammals and can cause periodontitis,pulp necrosis,chorioamnionitis,premature birth,neonatal septicemia,abscess,colorectal cancer and other infectious diseases.At present,the detection methods for F.nucleatum mainly include the isolation and culture and identification of bacteria,enzyme linked immunosorbent assay（ELISA）,polymerase chain reaction（PCR）,quantitative realtime PCR technology（qPCR）,etc.However,qPCR depends PCR equipment,skilled technician and clean operation environment.Therefore,a fast,simple and highly sensitive F.nucleatum assay is urgently needed for detection of F.nucleatum in clinic.In this study,magnetic nanoparticles（MNP）probe-based dark-field（DF）count method was established to detect F.nucleatum in samples rapidly and sensitively.This study mainly consists of the following parts:1.Preparation and identification of polyclonal antibodies against F.nucleatumBALB/c mice were immunized with F.nucleatum inactivated by formaldehyde as an antigen to prepare a serum containing F.nucleatum polyclonal antibody.The serum titer was confirmed to be 1:128000 by ELISA,and the purification effect was verified by SDS-PAGE after serum purification.The results showed that non-target protein bands decreased,which proved that the purification effect was good.The purified polyclonal antibody was confirmed to bind specifically to F.nucleatum by ELISA.2.Establishment of qPCR for quantitative detection of F.nucleatumSpecific primers for qPCR were designed and synthesized based on the conserved F.nucleatum specific gene nusG.Recombinant plasmids containing the target gene were constructed as standard plasmids.The genomes of F.nucleatum and other bacterial genomes were used as templates for qPCR reaction,the results showed that only F.nucleatum genome had target gene amplification,indicating good specificity of the primers.The standard curve of qPCR reaction was drawn with standard plasmids of gradient dilution.The equation was Y=-3.373X+42.77（Y was Ct value,X was the logarithm of plasmid copy number）,and the correlation coefficient R2=0.9982,indicating that the standard curve had a good linear relationship.The established qPCR method was used for quantitative of F.nucleatum was 1.71×102 copies/μL and the detection time was about 2 h.3.Establishment of the MNP probe based count with dark field microscope for quantitative detection of F.nucleatum（1）Preparation and characterization of MNP probe:MNP probe that can specifically capture F.nucleatum were prepared by coupling anti-F.nucleatum polyclonal antibodies with magnetic nanoparticles（MNPs）of 50 nm size and surface modified with Protein G.The results of SDS-PAGE and Transmission electron microscope（TEM）showed that MNP probe was successfully coupled with the antibody and the monodispersity was good,which confirmed that the probe was successfully prepared.（2）Detection of F.nucleatum with MNP probe-based dark-field microscopy:F.nucleatum and MNP probe were incubated together,and bright fusiform structures were observed under the dark field microscope,significantly different from background and negative controls.TEM observations showed that the MNP probe only bound F.nucleatum tightly but did not bind negative bacteria,indicating that the MNP probe could only specifically capture F.nucleatum.The bright fusiform structure was formed by the binding of the MNP probe on the surface of F.nucleatum.The MNP probe-based DF count method was used for quantitative detection of F.nucleatum.and the results were consistent with those of qPCR.The detection limit was 3.14×101 copies/μL,and the detection time was about 30 min.By comparison,MNP probe-based DF count method is about 5 times more sensitive than that of qPCR method,and the detection time is only a third that of qPCR.To demonstrate the accuracy and sensitivity of this method for F.nucleatum in real samples,C57BL/6J mice infected with F.nucleatum by enema,and colorectal tissue were collected their as real samples.F.nucleatum in samples was quantified by MNP probe-based DF count method and qPCR.The results show that the detection results of the two methods are consistent,but MNP probebased DF count method has a shorter detection time of 30 min.In conclusion,MNP probe-based DF count method for detection of F.nucleatum is simple,rapid（about 30 min）,sensitive and can be used for practical detection,providing a new technique for ultra-sensitive detection of F.nucleatum.It has a prospect of application for clinical samples detection.