PCRmax Legionella (all species) Instruction Manual

23S/5S Intergenic Spacer Region

Legionella_spp (all
species)

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Legionella is a genus of Gram-negative bacterium. This genus contains over 50 species, of
which there are 2 which cause pathogenesis in humans; these being L. pneumophila and
L. longbeachae. All of these bacteria can cause Legionellosis, which can be a relatively
common cause of nosocomial and community acquired pneumonia.
Legionella pneumophila is a flagellated bacterium which is a non-encapsulated, aerobic
bacillus of approximately 2um in length with a single, polar flagellum. The genome of this
bacterium is over 3Mbp long and encodes for around 3000 genes. There are several
serotypes of L. pneumophila, of which serotype 1 is responsible for 70-90% of cases.
The natural environment of L. pneumophila is hot and moist with a supply of nutrition from
the sediment. These environmental features can be found in storage facilities for drinking
water leading to infection of individuals by inhalation. After inhalation L. Pneumophila avoid
the defenses of the upper respiratory tract and target the alveolar. Here, macrophages and
epithelial cells can be infected by the bacteria via phagocytosis. The generated
phagosome does not fuse with the lysosome and so the bacteria avoid destruction. After
several hours within the host cell, endosome enclosure allows bacterial multiplication. The
bacteria express genes known to be involved in necrosis and exit the host cell and spread
intracellularly.

L. pneumophila infection is known as legionellosis which can cause Legionnaires' disease
or Pontiac fever. Legionnaires’ disease initially presents in fever, headache, aching joints,
lack of energy or tiredness that can progress into, cough, diarrhea, nausea, abdominal
pain and eventually pneumonia, which can lead to coma and death. Pontiac Fever can
cause symptoms including fever, headache, tiredness, loss of appetite, muscle and joint
pain, nausea, coughing but usually desists within 5 days without medical treatment.
Legionella micdadei is the second most commonly isolated Legionella species and is
responsible for approximately 60% of Legionella pneumonias other than those caused by
L. pneumophila. The pneumonia caused is a variant of Legionnaires’ disease called
Pittsburgh Pneumonia and is thought to occur mainly in immunocompromised patients.
The natural environment of Legionella longbeachae is in damp soil and is known to infect
humans via inhalation of particles from contaminated soil or compost. This bacterium is
thought to cause pneumonia in people with diseases of the immune system.

Introduction to Legionella_spp (all species)

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MIN

MAX

The 23S/5S intergenic spacer gene, is the ideal target to achieve a broad based detection
profile within this family. 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 Legionella_spp (all species) (Legionella_spp) genomes is
designed for the in vitro quantification of Legionella_spp genomes. The kit is designed to
have the broadest detection profile possible whilst remaining specific to the
Legionella_spp genome.

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

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

FAM labelled

• Legionella_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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