Development and Application of an Immunocapture PCR Diagnostic Assay Based on the Monoclonal Antibody for the Detection of Shigella

Document Type: Research Paper

Authors

1 Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR China.

2 Kunming Biomed International, Kunmimg 650500, China.

3 Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, 727 Jingming South Road, Kunming 650500, P.R. 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 specifc 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 afnity and specifcity 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 artifcial milk. The accuracy of the IC-PCR was confrmed 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.

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