PCRmax Corynebacterium diptheriae toxin A and B (active) Instruction Manual

C.diphtheriae Toxin sub-units A &
B

Corynebacterium
diphtheriae A and B

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

1

Quantification of Corynebacterium diphtheriae A and B genomes.

Advanced kit handbook HB10.07.08

Published Date: 13/05/2016

Corynebacterium diptheriae is a rod-shaped, non-motile, Gram-positive bacterium of the
Corynebacteriaceae family. This bacterium ranges in size from 1-8µm in length a genome
of around 2.5M nucleotides arranged in a single circular formation. This bacterium is
pathogenic to humans and causes the disease diphtheria.

C. diphtheria resides in the nose, mouth, throat and skin of humans and causes infections
that can be classified as two different forms of Diphtheria: respiratory and cutaneous, with
cutaneous diphtheria resulting in sores and ulcers on the skin and the respiratory disease
causing an infection of the upper respiratory tract by adhering to the pharyngeal cells and
colonizing them. This bacterial adhesion results in a pseudomembrane forming over the
cells which develops into lesions. C. diphtheria infections may become toxigenic when the
bacteria produce Diphtheria toxin although to produce the exotoxin extracellular levels of
iron must be low and the bacteria must be infected by a bacteriophage which is encodes
the tox gene.

Diphtheria toxin consists of 2 domains, an active domain and a binding domain. The toxin
binds to receptors on the host cell and is internalized by receptor mediated endocytosis.
Once within the endosome, the toxin undergoes a conformational change initiated by the
acidity of the endosome that allows the active domain to insert into the endosome
membrane. The binding domain is then cleaved allowing the active domain to be released
into the cytoplasm where it intereferes with protein synthesis by deactivating Elongation
factor II. This ultimately leads to cell death.

Cutaneous diphtheria infection shows symptoms after an incubation period of around 7
days. The respiratory condition has a shorter incubation period of up to 5 days and usually
presents with sore throat, fatigue and fever. In around 10% of cases, the infection can lead
to a swollen neck that causes breathing difficulties or asphyxiation. More generalized
symptoms may be listlessness or a fast heart rate which may lead to cardiomyopathy or
peripheral neuropathy. Transmission of this bacterium is mainly via aerosol secretions of
infected individuals and so the disease is very contagious but the infection can be treated
with antibiotics.

Introduction to Corynebacterium diphtheriae A and B

2

Quantification of Corynebacterium diphtheriae A and B genomes.

Advanced kit handbook HB10.07.08

Published Date: 13/05/2016

MIN

MAX

The target sequences (toxin subunits A and B) have previously been shown to be a good
genetic marker for C. diphtheriae in other real time PCR based studies (Mothershed EA
et.al 2002). The primers and probe sequences in this kit have 100% homology with a
broad range of clinically relevant reference sequences based on a comprehensive
bioinformatics analysis.

The PCRmax qPCR Kit for Corynebacterium diphtheriae A and B (C.diphtheriae A and B)
genomes is designed for the in vitro quantification of C.diphtheriae A and B genomes. The
kit is designed to have the broadest detection profile possible whilst remaining specific to
the C.diphtheriae A and B genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
C.diphtheriae A and B 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

3

Quantification of Corynebacterium diphtheriae A and B genomes.

Advanced kit handbook HB10.07.08

Published Date: 13/05/2016

Kit Contents

• Corynebacterium diphtheriae A primer/probe mix (150 reactions BROWN)

FAM labelled

• Corynebacterium diphtheriae B primer/probe mix (150 reactions BROWN)

FAM labelled

• Corynebacterium diphtheriae A positive control template (for Standard curve RED)

• Corynebacterium diphtheriae B 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 templates 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

4

Quantification of Corynebacterium diphtheriae A and B genomes.

Advanced kit handbook HB10.07.08

Published Date: 13/05/2016