Abstract
In this study, we describe a DNA microarray assay by using bead-mediated visible light-assisted signal detection for simultaneous screening of seven clinically important enteric pathogens, including Escherichia coli O157:H7, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella spp., Staphylococcus aureus, Rotavirus, and Norwalk virus (including genogroup I and II). Seven pairs of primers, in which the forward primers were labeled with biotin at the 5′ end, were designed and two sets of multiplex asymmetric PCR system were established to amplify the target genes of the seven pathogens. Twelve type specific oligonucleotides were designed and immobilized onto the aldehyde radical modified glass slide to function as target capture probes. After hybridization and stringency washes, the hybridized biotinylated PCR products were detected by the streptavidin-coated magnetic beads. The final hybridization results were visible to the naked eyes and can be imaged by CCD or digital camera. A total of 86 samples previously identified by conventional microbiological methods and/or PCR method were randomly selected to assess the specificity of this assay by a blind study. A coincidence rate of 100% was obtained. Due to the simplicity and specificity of the magnetic bead based DNA microarray, it is especially appropriate for the diagnosis and monitoring of enteric infectious diseases in the community and seaport.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Blackstone GM, Nordstrom JL, Vickery MC, Bowen MD, Meyer RF, DePaola A (2003) Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR. J Microbiol Methods 53:149–155
Guerrant RL, Lima AA, Barboza M, Young S, Silva T, Barrett L, Bao Y, Fox J, Moore S (1999) Mechanisms and impact of enteric infections. Adv Exp Med Biol 473:103–112
Hoshino K, Yamasaki S, Mukhopadhyay AK, Chakraborty S, Basu A, Bhattacharya SK, Nair GB, Shimada T, Takeda Y (1998) Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunol Med Microbiol 20:201–207
Jääskeläinen AJ, Maunula L (2006) Applicability of microarray technique for the detection of noro- and astroviruses. J Virol Methods 136:210–216
Jin D, Qi H, Chen S, Zeng T, Liu Q, Wang S (2008) Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification. J Microbiol Methods 75:365–368
Kostrzynska M, Bachand A (2006) Application of DNA microarray technology for detection, identification, and characterization of food-borne pathogens. Can J Microbiol 52:1–8
Leblanc N, Gantelius J, Schwenk JM, Ståhl K, Blomberg J, Andersson-Svahn H, Belák S (2009) Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses. J Virol Methods 155:1–9
Li CX, Pan Q, Guo YG, Li Y, Gao HF, Zhang D, Hu H, Xing WL, Mitchelson K, Xia K, Dai P, Cheng J (2008) Construction of a multiplex allele-specific PCR-based universal array (ASPUA) and its application to hearing loss screening. Hum Mutat 29:306–314
Logan C, O’Leary JJ, O’Sullivan N (2007) Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis. J Virol Methods 146:36–44
Mao Y, Zhu C, Boedeker EC (2003) Foodborne enteric infections. Curr Opin Gastroenterol 19:11–22
Mao Z, Zheng H, Wang X, Lin S, Sun Y, Jiang B (2008) DNA microarray for direct identification of bacterial pathogens in human stool samples. Digestion 78:131–138
Petri WA Jr, Miller M, Binder HJ, Levine MM, Dillingham R, Guerrant RL (2008) Enteric infections, diarrhea, their impact on function, development. J Clin Invest 118:1277–1290
Pettersson E, Ståhl PL, Mahdessian H, Käller M, Lundeberg J, Ahmadian A (2009) Visual DNA as a diagnostic tool. Electrophoresis 30:3691–3695
Ståhl PL, Gantelius J, Natanaelsson C, Ahmadian A, Andersson-Svahn H, Lundeberg J (2007) Visual DNA—identification of DNA sequence variations by bead trapping. Genomics 90:741–745
Taton TA, Mirkin CA, Letsinger RL (2000) Scanometric DNA array detection with nanoparticle probes. Science 289:1757–1760
Toranzos GA, Alvarez AJ (1992) Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters. Can J Microbiol 38:365–369
Wilhelmi I, Roman E, Sánchez-Fauquier A (2003) Viruses causing gastroenteritis. Clin Microbiol Infect 9:247–262
You Y, Fu C, Zeng X, Fang D, Yan X, Sun B, Xiao D, Zhang J (2008) A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea. J Microbiol Methods 75:566–571
Acknowledgments
This work was supported by funding from General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (2005IK089), and by Natural Science Foundation Project of CQ CSTC2007BB5080.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Sun, H., Mo, QH., Lin, JC. et al. Rapid simultaneous screening of seven clinically important enteric pathogens using a magnetic bead based DNA microarray. World J Microbiol Biotechnol 27, 163–169 (2011). https://doi.org/10.1007/s11274-010-0442-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-010-0442-3