PCRmax Shiga toxin (stx2A) producing Escherichia coli Instruction Manual

E.coli generic detection of beta­D-glucuronidase (uidA) gene
&
shiga toxin (STX2A) gene

shiga toxin (STX2A)
producing Escherichia
coli

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Escherichia coli are 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 bacteria are Gram-negative, rod-shaped, flagellated and non-spore forming.
Most strains are non-pathogenic but some cause food poisoning in humans with
transmission largely being through the faecal-oral route. E.coli have a circular, DNA
genome of approximately 4.6 Mb but also carry plasmids.

Shiga toxin-producing E. coli (STEC) are a form of enterohaemorrhagic E.coli that cause
illness ranging from mild intestinal disease to severe kidney disease. The shiga toxin can
cause haemorrhagic colitis, the source of the bloody diarrhoea associated with E. coli
O157:H7 infections, as well as being responsible for haemolytic uremic syndrome (HUS).
Shiga toxins derive their name from the organism where they were first classified, Shigella
dysenteriae. When the shiga toxin is released, it can translocate to organs other than the
digestive tract such as the kidneys and central nervous system. The ability of the shiga
toxins to pass through cell barriers is possibly due to the increased permeability of the
intestinal epithelial cells resulting from effects of the body’s own immune system. The body
increases permeability of cell barriers so that important cells of the immune system
(neutrophils/PMN’s) can reach the E. coli infection. Shiga toxin may use this opportunity to
break through the walls of the digestive tract, enter the blood stream, and bind white blood
cells for transport to locations such as the kidney or brain.

Enterohaemorrhagic E. coli are found in humans, cattle, and goats. There are a number of
E. coli serogroups that produce shiga toxin such as O157:H7, O26, O111, and O103.
Typical symptoms include severe abdominal cramping, sudden onset of watery diarrhoea,
frequently bloody, and sometimes vomiting and a low-grade fever. Most often the illness is
mild and self-limited generally lasting 1-3 days. However, serious complications such as
haemorrhagic colitis, haemolytic uremic syndrome (HUS), or post diarrhoeal thrombotic
thrombocytopenic purpura (TTP) can occur in up to 10% of cases. The incubation period
ranges from 1 to 8 days, and transmission is predominantly through consumption of
contaminated foods.

Introduction to shiga toxin (STX2A) producing
Escherichia coli

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MIN

MAX

This kit detects stx2A variations a,b,c,d,e, and g.
This kit does not detect stx2A variation f. This f subtype is not believed to be relevant to
human disease.
The generic E.coli portion of this kit also detects:
Shigella sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii, Rhizobium, Carica
papaya, Arabidopsis thaliana, Phytophthora capsici.

The PCR Max qPCR Kit for shiga toxin (STX2A) producing Escherichia coli (E.
coli_STX2A ) genomes is designed for the in vitro quantification of E.coli_STX2A
genomes. The kit is designed to have the broadest detection profile possible whilst
remaining specific to the E.coli_STX2A genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
E.coli_STX2A 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_STX2A specific primer/probe mix (150 reactions BROWN)

FAM labelled

• E.coli_STX2A 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 is 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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