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Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces.
Foodborne Pathog Dis. 2012 Jun; 9(6):541-8.FP

Abstract

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10⁵ colony-forming units (CFU)/g before enrichment and 10² CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.

Authors+Show Affiliations

Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas 66506-5606, USA. jbai@vet.k-state.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Evaluation Study
Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

22568751

Citation

Bai, Jianfa, et al. "Applicability of a Multiplex PCR to Detect the Seven Major Shiga Toxin-producing Escherichia Coli Based On Genes That Code for Serogroup-specific O-antigens and Major Virulence Factors in Cattle Feces." Foodborne Pathogens and Disease, vol. 9, no. 6, 2012, pp. 541-8.
Bai J, Paddock ZD, Shi X, et al. Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Pathog Dis. 2012;9(6):541-8.
Bai, J., Paddock, Z. D., Shi, X., Li, S., An, B., & Nagaraja, T. G. (2012). Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. Foodborne Pathogens and Disease, 9(6), 541-8. https://doi.org/10.1089/fpd.2011.1082
Bai J, et al. Applicability of a Multiplex PCR to Detect the Seven Major Shiga Toxin-producing Escherichia Coli Based On Genes That Code for Serogroup-specific O-antigens and Major Virulence Factors in Cattle Feces. Foodborne Pathog Dis. 2012;9(6):541-8. PubMed PMID: 22568751.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces. AU - Bai,Jianfa, AU - Paddock,Zachary D, AU - Shi,Xiaorong, AU - Li,Shubo, AU - An,Baoyan, AU - Nagaraja,Tiruvoor G, Y1 - 2012/05/08/ PY - 2012/5/10/entrez PY - 2012/5/10/pubmed PY - 2012/10/10/medline SP - 541 EP - 8 JF - Foodborne pathogens and disease JO - Foodborne Pathog Dis VL - 9 IS - 6 N2 - An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10⁵ colony-forming units (CFU)/g before enrichment and 10² CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods. SN - 1556-7125 UR - https://www.unboundmedicine.com/medline/citation/22568751/Applicability_of_a_multiplex_PCR_to_detect_the_seven_major_Shiga_toxin_producing_Escherichia_coli_based_on_genes_that_code_for_serogroup_specific_O_antigens_and_major_virulence_factors_in_cattle_feces_ L2 - https://www.liebertpub.com/doi/10.1089/fpd.2011.1082?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -