PCRmax Escherichia coli 0157:H7 Instruction Manual

tir (translocated intimin receptor)

Escherichia coli 0157:
H7

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Escherichia coli is one of many species of bacteria living in the lower intestines of
mammals, known as gut flora. When located in the large intestine, it assists with waste
processing, vitamin K production, and food absorption. Discovered in 1885 by Theodor
Escherich, a German pediatrician and bacteriologist, E. coli are abundant: the number of
individual E. coli bacteria in the faeces that a human defecates in one day averages
between 100 billion and 10 trillion. However, the bacteria are not confined to the
environment, and specimens have also been located, for example, on the edge of hot
springs. The E. coli strain O157:H7 is one of hundreds of strains of the bacterium that
causes illness in humans.

E. coli are unable to sporulate. Thus, treatments which kill all active bacteria, such as
pasteurization or simple boiling, are effective for their eradication, without requiring the
more rigorous sterilization which also deactivates spores.

As a result of their adaptation to mammalian intestines, E. coli grow best in vivo or at the
higher temperatures characteristic of such an environment, rather than the cooler
temperatures found in soil and other environments.

The enteric E. coli (EC) are divided on the basis of virulence properties into enterotoxigenic
(ETEC – causative agent of diarrhea in humans, pigs, sheep, goats, cattle, dogs, and
horses), enteropathogenic (EPEC – causative agent of diarrhea in humans, rabbits, dogs,
cats and horses); enteroinvasive (EIEC – found only in humans), verotoxigenic (VTEC –
found in pigs, cattle, dogs and cats); enterohaemorrhagic (EHEC – found in humans,
cattle, and goats, attacking porcine strains that colonize the gut in a manner similar to
human EPEC strains) and enteroaggregative E. coli (EAggEC – found only in humans).

E. coli O157:H7 was first recognized as a pathogen as a result of an outbreak of unusual
gastrointestinal illness in 1982. The outbreak was traced to contaminated hamburgers, and
the illness was similar to other incidents in the United States and Japan. The etiologic
agent of the illness was identified as a rare O157:H7 serotype of Escherichia coli in 1983.
This serotype had only been isolated once before, from a sick patient in 1975.

Introduction to Escherichia coli 0157:H7

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MIN

MAX

The primers have 100% homology with all reference sequences included in the NCBI
database and list below. They therefore have a very broad quantification profile.

DQ458771.1, BA000007.2, AF070067.1 AF125993.1, AF071034.1, AE005174.2,
DQ007021.1, DQ007020.1, DQ007019.1, AB355659.1, AY944737.1, AB288104.1,
AJ303141.2, AJ277443.1, AJ223063.1, AF453441.1, AF200363.1, AY944738.1,
AY944736.1, AY944735.1, AF113597.1, AF070069.1, AF070068.1, AF132728.1,
U59504.2, U59502.2, AF045568.1, U59503.1

The PCR Max qPCR Kit for Escherichia coli 0157:H7 (E.coli_0157) genomes is designed
for the in vitro quantification of E.coli_0157 genomes. The kit is designed to have the
broadest detection profile possible whilst remaining specific to the E.coli_0157 genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
E.coli_0157 sequences based on a comprehensive bioinformatics analysis.

If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to [e-mail] and our bioinformatics team will
answer your question.

Specificity

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Kit Contents

• E.coli_0157 specific primer/probe mix (150 reactions BROWN)

FAM labelled

• E.coli_0157 positive control template (for Standard curve RED)

• Internal extraction control primer/probe mix (150 reactions BROWN)

VIC labelled as standard

• Internal extraction control DNA (150 reactions BLUE)

• Endogenous control primer/probe mix (150 reactions BROWN)

FAM labelled

• RNAse/DNAse free water (WHITE)

for resuspension of primer/probe mixes and internal extraction control DNA

• Template preparation buffer (YELLOW)

for resuspension of positive control template and standard curve preparation

Reagents and equipment to be supplied by the user

Real-Time PCR Instrument

DNA extraction kit

This kit designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.

Lyophilised 2x qPCR Mastermix

This kit is designed to be compatible with all commercially available Mastermixes that run
with standard cycling conditions.

Pipettors and Tips

Vortex and centrifuge

Thin walled 1.5 ml PCR reaction tubes

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