Author/Authors :
Zhang, Liding Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Du, Xuewei Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Wei, Qiujiang Kunming Biomed International - Kunming 650500, China , Han, Qinqin Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Chen, Qiang Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Zhang, Mi Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Xia, Xueshan Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Song, Yuzhu Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China , Zhang, Jinyang Research Center of Molecular Medicine of Yunnan Province - Faculty of Life Science and Technology - Kunming University of Science and Technology, Kunming 650500, PR China
Abstract :
Background: Shigella is among the most important human pathogenic microorganisms, infecting both humans and nonhuman and causing clinically severe diarrhea. Shigella must be enriched before detection, which is time-consuming.
Objectives: To develop a sensitive, rapid, and specific method for Shigella detection.
Materials and Methods: Shigella was used as an antigen to generate monoclonal antibodies (mAbs). mAbs were screened via indirect enzyme-linked immunosorbent assay (ELISA) and western blot, and two mAbs were selected. The mAb A3 showed high affinity and specificity and was used to develop immune magnetic beads (IMBs) for Shigella enrichment. An immunocapture (IC)-PCR primer was designed from the ipaH gene, and IC-PCR was developed based on the IMBs and PCR.
Results: This system shortened the Shigella detection time to 70 min. The sensitivity of the IC-PCR was 9 colony-forming units.mL-1 in artificial milk. The accuracy of the IC-PCR was confirmed using 46 clinical samples collected from monkeys. The IC-PCR results were consistent with the serological and biochemical assays.
Conclusion: The IC-PCR described herein accurately detected Shigella from milk samples, monkeys and can thus be used to complement classical detection methods.
Keywords :
Magnetic Immunocapture , Monoclonal Antibodies , PCR , Shigella
