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High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites
Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.
Author Notes
Financial Support: This work was supported by National Institutes of Health grants U01 AI075396, AI043596, and the Bill and Melinda Gates Foundation.
Authors' addresses: Mami Taniuchi, William A. Petri Jr., and Eric R. Houpt, Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, VA, E-mails: mt2f@virginia.edu, wap3g@virginia.edu, and erh6k@virginia.edu. Jaco J. Verweij and Lisette van Lieshout, Departments of Parasitology and Medical Microbiology (Clinical Microbiology Laboratory), Leiden University Medical Center, Leiden, The Netherlands, E-mails: J.J.Verweij@lumc.nl and LvanLieshout@lumc.nl. Zannatun Noor, Shihab U. Sobuz, and Rashidul Haque, International Centre for Diarrhoeal Disease Research (ICDDR,B), Centre for Health and Population Research, Mohakhali, Dhaka, Bangladesh, E-mails: zn5qh@virginia.edu, shihab119@icddrb.org, and rhaque@icddrb.org.
Reprint requests: Mami Taniuchi, Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, 345 Crispell Dr., MR6 Room 1714, Charlottesville, VA 22908, Tel: 434-924-5575, Fax: 434-924-0075, E-mail: mt2f@virginia.edu.