1948 Journal o f Food Protection, Vol. 76, No. 11, 2013, Pages 1948-1957 doi: 10.4315/0362-028X.JFP-13-153 Copyright © , International Association for Food Protection

Research Note

Customizable PCR-Microplate Array for Differential Identification of Multiple Pathogens ABDELA WOUBIT,* TESHOME YEHUALAESHET, SHERRELLE ROBERTS, MARTHA GRAHAM, MOONIL KIM,+ TEMESGEN SAMUEL*

AND

Department o f Pathobiology, College o f Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, Alabama 36088, USA MS 13-153: Received 15 April 2013/Accepted 8 July 2013

ABSTRACT Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shigella dysenteriae, Escherichia coli 0157:H7, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. novicida, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, and Yersinia pseudotuber­ culosis. Previously, we identified highly specific primers targeting each of these pathogens. Here, we report the development of customizable PCR-microplate arrays for simultaneous identification of the pathogens using the primers identified. A mixed aliquot of genomic DNA from 38 strains was used to validate three PCR-microplate array formats. Identical PCR conditions were used to run all the samples on the three formats. Specific amplifications were obtained on all three custom plates. In preliminary tests performed to evaluate the sensitivity of these assays in samples inoculated in the laboratory with Salmonella Typhimurium, amplifications were obtained from 1 g of beef hot dog inoculated at as low as 9 CFU/ml or from milk inoculated at as low as 78 CFU/ml. Such microplate arrays could be valuable tools for initial identification or secondary confirmation of contamination by these pathogens.

Foodbome illnesses cause serious economic and public health burdens worldwide. According to recent reports, 48 million Americans suffer annually from domestically acquired foodbome illness associated with 31 identified pathogens and a broad category of unspecified agents (1921). The potential impact of deliberate or accidental adulteration of food by organisms such as Francisella tularensis, Yersinia pestis, and Bacillus anthracis is difficult to estimate. Many documented examples of unintentional foodbome outbreaks that have sickened thousands of people and killed hundreds provide a grim basis for estimating the impact of deliberate food adulteration (2, 22). In the United States, Salmonella alone causes over 1 million cases of illness annually, leading to over 19,000 estimated hospitalizations and 380 deaths (20). The reported incidence of Shigella infections was 2,848 cases per 100,000 population in 2007 (3). The Centers for Disease Control and Prevention (CDC) (3) estimated that among Shigella species, S. sonnei accounts for approximately 78% of all isolates. Compared with data from 2006 to 2008, the incidence of pathogen-specific illness in 2010 was lower for Shiga toxin-producing Escherichia coli 0157 and Shigella infections but higher for non-0157 E. coli infections, mostly * Authors for correspondence. Tel: 334-724-4547; Fax: 334-724-4110; E-mail: [email protected] (T. Samuel). Tel: 334-727-8278; Fax: 334-724-4110; E-mail: [email protected] (A. Woubit). f Present address: BioNanotechnology Research Center (BNRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea.

026 (37%), 0103 (24%), and O il 1 (17%) strains. Among 186 laboratory-confirmed Vibrio infections, V. parahaemo­ lyticus (57%) and V. vulnificus (13%) were the most prevalent (3). Fast and accurate identification of microbial pathogens in food samples by public health agencies and diagnostic laboratories could enable timely detection and intervention measures during outbreaks. Real-time PCR is one of the principal methodologies for rapid diagnosis of foodbome outbreaks (6). Multiplexed simultaneous detection of pathogens by using real-time PCR is one of the approaches used to identify multiple pathogens during an outbreak. However, the number of pathogens detectable by multiplex methodologies has been limited by the cost to run such assays and by technical or instrumentation capacity limits. A customizable multiplex assay platform would be beneficial by limiting the number of tests needed based on a presumptive diagnosis or the likelihood of infection or contamination by certain pathogens. For example, a multiplex detection method could be customized to perform simultaneous screening for selected foodbome pathogens, including enteric and toxin-producing bacteria, or to selectively detect pathogens of biothreat potential. In this study, we developed customizable 96-well PCRmicroplate arrays for simultaneous detection of 12 patho­ gens, which were selected based on their economic significance as foodbome pathogens, their potential as biothreat food contaminants, or in the cases of Yersinia pseudotuberculosis and Francisella novicida, as surrogates

J. Food Plot., Vol. 76, No. 11

DETECTION OF MULTIPLE PATHOGENS BY PCR-MICROPLATE ARRAY

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TABLE 1. Lists of organisms used in the validation of customizable PCR-micropiate arrays Species, strains

CDC category"

Escherichia E. coli 0157:H7 EDL933 E. coli 1175 E. coli 35334 Francisella F. tularensis F. tularensis F. tularensis F. tularensis

subsp. tularensis Schu S4 subsp. novicida U112 subsp. novicida KM 145 subsp. philomiragia

Origin*

B

ATCC ATCC ATCC

A

Dr. Karl Klose Dr. Karl Klose BEI ATCC

Salmonella Salmonella Braenderup ATCC (BAA-664) Salmonella Typhimurium LT2 ATCC (700720D-5) Salmonella Typhimurium ATCC51812 Salmonella Typhimurium Strain ATCC 700730 Salmonella Typhimurium Strain MZ1589 ATCC BAA-1836 S. enterica subsp. enterica ATCC 11511 Salmonella Enteritidis var. Danysz ATCC 49216 Salmonella Typhi ATCC 39926 Salmonella Typhi Ty2 ATCC (700931) Salmonella Typhi ATCC 6539 Salmonella Saintpaul 127 ATCC (9712)

B B B B

ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC

Shigella S. dysenteriae ATCC (11456a) S. sonnei ATCC (11060)

B B

ATCC ATCC

B

ATCC ATCC ATCC ATCC

A A A A A

BEI BEI ATCC ATCC ATCC ATCC ATCC BEI (NR-804) ATCC ATCC BEI (NR-204) ATCC ATCC

B

Vibrio V. cholerae ATCC (39315) V. cholerae serogroup 01, serotype Ogawa, biogroup El Tor (ATCC BAA-1508) V. parahaemolyticus EB 101 ATCC (17802) V. vulnificus type strain, biogroup 1 ATCC (27562) Yersinia Y. pestis A 1122 BEI (NR-15) Y. pestis KIM 10+ BEI (NR-642) Y. pestis ZE 94-2111 Y. pestis Yokohama (ATCC BAA-1508) Y. pestis K25; D21 (ATCC BAA-1511) Y. pseudotuberculosis ATCC 11960 Y. pseudotuberculosis ATCC 908 Y. pseudotuberculosis P62 ATCC (29910) Y. pseudotuberculosis NCTC 10275 ATCC (29833) Y. enterocolitica subsp. enterocolitica ATCC 700823 T. enterocolitica Billups-1803-68 ATCC (23715) Y. enterocolitica WA ATCC (27729) Y. kristensenii ATCC 35669

B B

" The CDC lists biothreat agents as category A or category B. ATCC, American Type Culture Collection, Manassas, VA; BEI, BEI Resources, Manassas, VA. Dr. Karl Klose is from the University of Texas, San Antonio, and the South Texas Center for Emerging Infectious Diseases, San Antonio. for highly pathogenic strains. These microplate arrays can be used as specific tools for monitoring foodbome outbreaks. M ATERIALS A N D M E TH O D S Bacterial species, DNA sources, and primers. Most of the bacterial strains used in this study, their growth conditions, and DNA sources or preparations are described elsewhere (28). The complete list of bacterial species and strains, including 15 new strains included in the current study, is shown in Table 1. The

quality of all DNA specimens was assessed spectrophotometrically using the ND-1000 method (Nanodrop Technologies, Inc., Wilmington, DE) and by agarose gel electrophoresis. An A2m / A2go ratio of >1.8 was accepted as DNA of good quality. Detailed descriptions of the genome data mining, text mining, and comparative genome analysis that preceded localization of specific target regions for the eventual design of primers, including in silico validation and in vitro testing of selected primers, also are given elsewhere (28). The primers used are listed in Table 2. The validation of these primers included comprehensive analyses using

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J. Food Prot., Vol. 76, No. 11

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Customizable PCR-microplate array for differential identification of multiple pathogens.

Customizable PCR-microplate arrays were developed for the rapid identification of Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Typhi, Shig...
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