PCRmax Alpha toxin producing Clostridium perfringens Instruction Manual

(cpa) gene

Alpha toxin producing
Clostridium
perfringens (formally
C.perfringens_spp)

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Clostridium perfringens is an anaerobic, spore-forming, non-motile, Gram positive, rod
shaped bacterium of the genus Clostridium. It has a circular DNA genome of approximately
3Mb containing 2660 protein coding regions, and also carries genes on plasmids. It is
widely distributed in the environment and foods and forms part of the normal gut flora in
both man and animals. C. perfringens persists in soil, sediments and areas subject to
human or animal faecal pollution. In the UK and the USA it is the third most common cause
of food poisoning, often being associated with large scale catering and is largely linked to
C. perfringens type A. In addition, C. perfringens has been widely recognized as being the
most important causal organism of gas gangrene which killed hundreds of thousands of
soldiers in World War 1 and can cause serious nosocomial infection post surgery.

C. perfringens induced food poisoning is mostly caused by the consumption of poorly
cooked meat and poultry whereas entry via wounds leads to the more serious gas
gangrene. C. perfringens produces at least 16 virulence factors, including 12 toxins.
Strains of C. perfringens are classified as 5 biotypes A – E depending on the differential
production of four major (i.e. lethal) exotoxins (alpha (phospholipase A), beta, epsilon and
iota) which give the cytotoxic and necrotic activity associated with infection. Clostridial
haemolysins and extracellular enzymes such as proteases, lipases, collagenase and
hyaluronidase contribute to the invasive process and cytotoxicity. In addition, strains of C.
perfringens may also produce a number of other toxins, including: neuraminidase and
enterotoxin (Cpe).

The most common symptoms of C. perfringens food poisoning are intense abdominal
cramps and diarrhoea which begin 8-22 hours after consumption of foods containing large
numbers of those C. perfringens bacteria capable of producing the food poisoning toxin.
The diagnosis of C. perfringens is typically done by the above mentioned symptoms.
Confirmation of the illness can be done by the detection of toxins or other molecular
methods for the detection of the pathogen. In contrast gas gangrene is a form of wet
gangrene or localised cellular death which is fatal unless rapidly treated. The hallmark of
gas gangrene is crepitation, a result of carbon dioxide and hydrogen accumulation as a
metabolic by product in necrotic tissues. Other symptoms may include fever, coma,
anxiety, severe pain and kidney failure. The incubation period is usually 1 to 4 days but
can vary from 3 hours to 6 weeks or longer. Gas gangrene is rare, with only 1,000-3,000
cases yearly in the U.S.

Introduction to Alpha toxin producing Clostridium
perfringens (formally C.perfringens_spp)

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MAX

MIN

Our kit for All pathogenic C.perfringens strains (C.perfringens_cpa) genomes is designed
for the in vitro quantification of C.perfringens strains that have the cpa toxin gene
conferring the ability to cause infection. The kit is designed to have the broadest detection
profile possible whilst remaining specific to the C.perfringens_cpa target gene. The
primers and probe sequences in this kit have 100% homology with a broad range of cpa
toxin 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

• C.perfringens_cpa specific primer/probe mix (150 reactions BROWN)

FAM labelled

• C.perfringens_cpa 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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