A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.
Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Mark S
MS
Rundell
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Maneesh
M
Pingle
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Sanchita
S
Das
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Aashiq
A
Hussain
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021; Groupe Haitien d'Étude du Sarcome de Kaposi et des Infections Opportunistes, Port-Au-Prince, Haiti.
Oksana
O
Ocheretina
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021; Groupe Haitien d'Étude du Sarcome de Kaposi et des Infections Opportunistes, Port-Au-Prince, Haiti.
Macarthur
M
Charles
Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Davise H
DH
Larone
Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY 11794.
Eric D
ED
Spitzer
Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, Box 62, New York, NY 10021.
Linnie
L
Golightly
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Box 62, New York, NY 10021. Electronic address: barany@med.cornell.edu.
Francis
F
Barany
D001419:Bacteria / Q000145:classification / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D005517:Foodborne Diseases / Q000175:diagnosis* / Q000382:microbiology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000382:microbiology; D017408:Guidelines as Topic; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D054579:National Institute of Allergy and Infectious Diseases (U.S.); D012680:Sensitivity and Specificity; D014481:United States
Enteric pathogens; Infectious disease; Molecular diagnostics; Multiplex