Development of a Multiplex Real-Time PCR Assay with Internal Amplification Control for the Detection of Shigella Species and Enteroinvasive Escherichia coli

Shigella species, particularly S. sonnei and S. flexneri, remain some of the leading bacterial etiological agents of gastrointestinal diseases in the United States and globally. The isolation and detection of these foodborne pathogens are critical for preventing the spread of disease and facilitating epidemiological investigations aimed at determining the source of a Shigella infection outbreak. A multiplex real-time PCR-based assay was developed that targets all four species of Shigella plus enteroinvasive Escherichia coli. The assay incorporates primers directed to the ipaH genes located on both the virulence plasmid and chromosome, the plasmid-encoded virulence gene mxiC, a mutated mxiC gene (mxiC::kan) that differentiates wild-type strains from a laboratory control strain, and an internal amplification control. More than 50 isolates of all four Shigella species were tested for inclusivity and specificity of the multiplex PCR assay, and more than 30 non-Shigella isolates were tested for exclusivity of the assay. The sensitivity of the assay was 1 to 3 CFU and 5 to 50 fg of target (total) DNA for the ipaH, mxiC, and mxiC::kan gene targets. The assay performed equally well and with no measurable inhibition in the Shigella target reactions when rinsates of several high-risk produce commodities (parsley, cilantro, alfalfa sprouts, and lettuce) were added to the reactions. This multiplex PCR assay is sensitive and specific and has the added dimension of discriminating all Shigella species from the positive control strain so that in any sample analysis other strains can be excluded as a source of contamination.

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Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis , Cryptosporidium parvum / Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2015.10.019 ◽

2016 ◽

Vol 22 (2) ◽

pp. 190.e1-190.e8 ◽

Cited By ~ 28

Author(s):

A. Laude ◽

S. Valot ◽

G. Desoubeaux ◽

N. Argy ◽

C. Nourrisson ◽

...

Keyword(s):

Literature Review ◽

Multiplex Pcr ◽

Real Time ◽

Cryptosporidium Parvum ◽

Real Time Pcr ◽

Giardia Intestinalis ◽

Pcr Assay ◽

Cryptosporidium Hominis ◽

Multiplex Pcr Assay ◽

Stool Samples

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Real-Time Multiplex PCR Assay and Melting Curve Analysis for Identifying Diarrheagenic Escherichia coli

Journal of Clinical Microbiology ◽

10.1128/jcm.02478-12 ◽

2013 ◽

Vol 51 (3) ◽

pp. 1031-1033 ◽

Cited By ~ 18

Author(s):

Tamara B. Souza ◽

Diego M. Lozer ◽

Sônia M. S. Kitagawa ◽

Liliana C. Spano ◽

Neusa P. Silva ◽

...

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Real Time ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Pcr Assay ◽

Diarrheagenic Escherichia Coli ◽

Multiplex Pcr Assay

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Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

Applied and Environmental Microbiology ◽

10.1128/aem.00460-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5840-5847 ◽

Cited By ~ 227

Author(s):

Jessica L. Nordstrom ◽

Michael C. L. Vickery ◽

George M. Blackstone ◽

Shelley L. Murray ◽

Angelo DePaola

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

The United States ◽

Internal Amplification Control ◽

Most Probable Number ◽

Specific Marker ◽

Pcr Assay ◽

Probable Number ◽

The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

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Fate ofEscherichia coliO157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

Canadian Journal of Microbiology ◽

10.1139/w04-097 ◽

2004 ◽

Vol 50 (12) ◽

pp. 1007-1014 ◽

Cited By ~ 55

Author(s):

A Mark Ibekwe ◽

Pamela M Watt ◽

Peter J Shouse ◽

Catherine M Grieve

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Irrigation Water ◽

Real Time Pcr ◽

The United States ◽

Large Animal ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli ◽

Rhizosphere Soils

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 × 103in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.Key words: real-time PCR, Escherichia coli O157:H7, irrigation, survival, quantification.

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Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2018.02.007 ◽

2018 ◽

Vol 24 (11) ◽

pp. 1205-1209 ◽

Cited By ~ 6

Author(s):

F. Morio ◽

S. Valot ◽

A. Laude ◽

G. Desoubeaux ◽

N. Argy ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Giardia Intestinalis ◽

Pcr Assay ◽

Entamoeba Dispar ◽

Multiplex Pcr Assay ◽

Stool Specimens ◽

Entamoeba Moshkovskii

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Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v1i2.21506 ◽

2016 ◽

Vol 1 (2) ◽

pp. 38-42 ◽

Cited By ~ 4

Author(s):

Khairun Nessa ◽

Dilruba Ahmed ◽

Johirul Islam ◽

FM Lutful Kabir ◽

M Anowar Hossain

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Clinical Laboratory ◽

Proteinase K ◽

Microbiology Laboratory ◽

Pcr Assay ◽

E Coli ◽

Diarrheagenic Escherichia Coli ◽

Multiplex Pcr Assay ◽

Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

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