)ORULGD6WDWH8QLYHUVLW\/LEUDULHV 2018 High-Resolution Melt Curve PCR Assay for Specific Detection of E. coli O157:H7 in Beef Yuejiao Liu, Prashant Singh and Azlin Mustapha This is the accepted manuscript, and the version of record can be found at https://doi.org/10.1016/j.foodcont.2017.11.025. Follow this and additional works at DigiNole: FSU's Digital Repository. For more information, please contact [email protected] 1 2 3 4 5 6 7 8 High-Resolution Melt Curve PCR Assay for Specific Detection of E. coli O157:H7 in Beef 9 Yuejiao Liu1, Prashant Singh2, Azlin Mustapha1* 10 11 Food Science Program, University of Missouri, Columbia1 12 Department of Nutrition, Food and Exercise Sciences, Florida State University2 13 2 14 15 16 17 18 19 20 21 *Corresponding author: Food Science Program, 246 William Stringer Wing, Eckles Hall, 22 University of Missouri, Columbia, MO 65211; Tel: (573) 882-2649, Fax: (573) 884-7964, E- 23 mail: [email protected] 1 24 Abstract 25 Among the disease-causing Shiga toxin-producing Escherichia coli (STEC), E. coli O157:H7 is 26 estimated to cause one-third of the total STEC illnesses and the most cases of hemolytic uremic 27 syndrome (HUS) in the U.S. The uidA gene which is present in the majority of E. coli strains, 28 codes for the synthesis of β-D-glucuronidase (GUD). In E. coli O157:H7, the uidA gene has a 29 single point mutation at the +93 position that leads to an alteration in the amino acid sequence 30 encoding the GUD enzyme. The aim of this study was to distinguish E. coli O157:H7 from other 31 E. coli using a high-resolution melt curve (HRM) real-time PCR assay. Based on the uidA 32 mutation in E. coli O157:H7, a reliable PCR assay targeting the uidA gene was developed to 33 differentiate E. coli O157:H7 from other E. coli serotypes and the closely related Shigella spp. 34 The assay was validated using a set of 129 pure bacterial DNA samples and spiked ground beef 35 and beef trim. Isolates of E. coli O157:H7 formed distinctive melt peaks that were easily 36 distinguishable from those of other E. coli serogroups and Shigella isolates in the PCR plot. 37 Therefore, this assay was able to clearly discriminate E. coli O157:H7 strains from other E. coli 38 and Shigella. With a 5-8 h enrichment time, 10 CFU of E. coli O157:H7 were detectable in 325 g 39 spiked beef samples. The HRM E. coli O157:H7 detection assay standardized in this study will 40 allow for accurate identification of contaminated food samples and help in preventing foodborne 41 outbreaks caused by this pathogen. 42 43 44 45 Keywords: E. coli O157:H7, ground beef, high resolution melt curve (HRM) PCR, 46 immunomagnetic separation, uidA 2 47 1. Introduction 48 Escherichia coli is a bacterium that is widely found in the intestinal tract of humans and 49 animals. Most E. coli strains are harmless, however some E. coli are pathogenic, among which, 50 Shiga toxin-producing E. coli (STEC) are of the most concern because of their strong association 51 with foodborne disease outbreaks. STEC typically cause mild diarrhea but can sometimes result 52 in severe illnesses, including hemolytic uremic syndrome (HUS) and bloody diarrhea (Tarr, 53 Gordon, & Chandler, 2005). STEC are reported to lead to numerous infections caused by 54 consumption of contaminated foods annually. 55 According to the CDC (2011), E. coli O157:H7 is responsible for over 73,000 illnesses in 56 the U.S. annually, resulting in around 2,000 hospitalizations and 60 deaths. Among disease- 57 causing STEC, E. coli O157:H7 is the predominant serotype that causes HUS in human. HUS is 58 a life-threatening disease characterized by acute renal failure and microvascular thrombi (Tarr, et 59 al., 2005). The management of HUS remains challenging since no specific treatments have been 60 developed. Therefore, the most effective way to prevent HUS thus far is to prevent the initial 61 infection with E. coli O157:H7. Because more than one-third of the total STEC illnesses and 62 outbreaks in the United States are caused by E. coli O157:H7 (Scallan, et al., 2011), 63 distinguishing this strain from other STEC is of critical importance for treating HUS. Further, 64 accurate detection of E. coli O157:H7 will help in pin-pointing its food source with the intention 65 of controlling foodborne outbreaks related to this dangerous pathogen. 66 Beef products and carcass were implicated to be the primary vehicle for enterohemorrhagic 67 E. coli O157:H7, and the transmission of E. coli O157:H7 usually happens during slaughter 68 processes. More than half of the E. coli O157:H7 infection cases were linked to foods derived 69 from cattle (Rangel, Sparling, Crowe, Griffin, & Swerdlow, 2005). In the U.S., E. coli O157:H7 3 70 is declared as an adulterant in ground beef. Therefore, mitigating and controlling this pathogen at 71 the initial stages of processing are crucial to decreasing the risk of E. coli O157:H7 infections. 72 More importantly, the minimum level of E. coli O157:H7 that can cause illness is very low, 73 usually less than several hundred CFU/g, necessitating a reliably selective detection method with 74 a high sensitivity (Karmali, 2004). 75 The traditional serotyping method is based on the immunological reaction of antigens 76 located on E. coli with specific antibodies. However, this method is time-consuming because it 77 involves sample enrichment, isolation of pure cultures using selective agar, biochemical testing 78 and serotyping. Further, culture-based methods have a low specificity since other bacteria, such 79 as Citrobacter freundii share the same antigens with E. coli O157:H7, resulting in cross- 80 reactions and false positive results (Bellisle, 1999; Vinogradov, Conlan, & Perry, 2000). 81 The 4-methylumbelliferyl-β-D-glucuronide (MUG) test is frequently used in identifying E. 82 coli (Feng & Hartman, 1982) since the majority of E. coli strains produces the enzyme, β-D- 83 glucuronidase (GUD). When MUG is hydrolyzed by GUD, a fluorogenic compound, 4- 84 methylumbelliferyl, is released which fluoresces when exposed to long-wave UV light (Feng, 85 1993). More than 90% of E. coli strains are positive in the MUG assay, while E. coli O157:H7 is 86 consistently negative for this test. The uidA gene controls the synthesis of GUD. Although E. 87 coli O157:H7 is MUG-negative, it contains the uidA gene that is highly identical to that of other 88 serotypes. The only difference is a base substitution in the sequence of the uidA gene. At the +93 89 position of its uidA gene sequence, E. coli O157 harbors a thiamine, while in other E. coli 90 serotypes, it is a guanine Feng, 1993). This leads to an alteration in the amino acid sequence that 91 encodes GUD enzyme. Therefore, this mutation is used as one of the most common targets for 92 PCR-based identification of the E. coli O157:H7 serotype. 4 93 Compared with culture-based methods, the Polymerase Chain Reaction (PCR) technique 94 which allows for rapid amplification of target genes in DNA isolates is widely used for 95 distinguishing bacterial species. The HRM-based PCR assays are highly sensitive to any single- 96 base change in amplified targets. HRM dyes are able to completely saturate amplicons, thus 97 yielding a melt curve with higher resolution. Owing to the simplicity, relatively low cost and 98 ease of use, HRM application in the areas of clinical diagnostics and food safety has also 99 increased. 100 Therefore, the objective of this study was to develop a HRM real-time PCR assay for 101 identification of E. coli O157:H7. This study will also provide a useful tool to help in 102 epidemiological analyses and control of foodborne outbreaks associated with this pathogen. 103 104 2. Materials and Methods 105 2.1 Bacterial DNA extraction 106 E. coli strains were obtained from the University of Missouri, Food Microbiology Lab 107 culture collection. Cultures were grown overnight at 37 oC in Tryptic Soy broth (TSB) (Difco 108 Labs, Sparks, MD, USA). Genomic DNA of pure E. coli strains was isolated from overnight 109 cultures in TSB broth. One milliliter of the overnight cultures in TSB broth was centrifuged at 110 6,000 ×g for 2 min. DNA from the obtained cell pellet was extracted by using PrepMan™ Ultra 111 Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) following the 112 manufacturer’s instructions. The concentration and purity of the obtained DNA samples were 113 measured by a Nanodrop Lite Spectrophotometer (Thermo Fisher, Wilmington, DE, USA). 5 114 Genomic DNA of E. coli from spiked beef samples was isolated using DNeasy® Blood and 115 Tissue Kit (Qiagen, Hilden, Germany), according to the instructions of the Purification of Total 116 DNA from Animal Tissues. 117 118 2.2 Primer and HRM-PCR assay design 119 The primer pair, HRM-F and HRM-R, was designed using the Primer3 software 120 (Untergasser et al., 2012). The specificity of the designed PCR primers was tested using 121 NCBI/Primer-BLAST, and all oligonucleotides were commercially synthesized (IDT, Coraville, 122 IA, USA) as shown in Table 1. 123 Real-time PCR was performed using 2× LightCycler® 480 High Resolution Melting Master 124 (Roche Diagnostics Corp., Indianapolis, USA), and the HRM assay was standardized on a 125 LightCycler® 96 real-time PCR instrument (Roche Diagnostics Corp., Indianapolis, USA). The 126 PCR reaction was performed in a 10 µL reaction volume in duplicate, with 30 ng of genomic 127 DNA, 0.5 µM primers and 2.5 mM MgCl2.