PCRmax Shigella (all species) Instruction Manual

virulence plasmid pCP301 (VirA)

Shigella species

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Shigella is a genus of four species of Gram-negative bacterium that is split into four
serogroups. Shigella species have genomes ranging in size from 4.4Mbp to 4.8Mbp in
length in a circular formation and are the causative agents of Shigellosis.

Shigella species infect the M cells of the intestinal epithelium and then releases proteins
into the host cell via the type-three secretion system that allows bacterial invasion. The
spread of bacteria between cells results in degradation of the epithelial lining of the
intestine resulting in leakage.

Infection with Shigella species results in symptoms such as fever, vomiting, abdominal
pain and diarrhoea, resulting from inflammation of the colon and intestinal lumen. Severe
symptoms can be treated with antibiotics and a course of rehydration therapy.

S. dysenteriae is the species of serogroup A and causes dysentery in many developing
countries. Serogroup B consists of serotypes of S.flexneri which is found in developing
countries and Serotype C contains S.boydi which is limited to the Indian subcontinent.
These 3 species are physiologically similar whereas S. sonnei of Serogroup D, found in
developed countries, is more genetically divergent.

Introduction to Shigella species

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MIN

MAX

The target sequence is within the virulence plasmid pCP301 (VirA) which is carried by
nearly all clinical isolates of shigella. This target has previously been shown to be a good
genetic marker for Shigella in other real time PCR based studies (Hiroshi Fukushima et.al

2003.) 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 PCR Max qPCR Kit for Shigella species (Shigella_spp) genomes is designed for the
in vitro quantification of Shigella_spp genomes. The kit is designed to have the broadest
detection profile possible whilst remaining specific to the Shigella_spp genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
Shigella_spp 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

• Shigella_spp specific primer/probe mix (150 reactions BROWN)

FAM labelled

• Shigella_spp 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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