PCRmax Legionella pneumophila Instruction Manual

Macrophage Infectivity
Potentiator (mip) Gene

Legionella
pneumophila

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Legionella pneumophila is a thin, aerobic, Gram-negative bacterium of the genus
Legionella. It is a facultative intracellular organism that is the causative agent of
legionellosis or Legionnaires' disease. This disease was first discovered in 1976 among a
group of elderly men attending an American legion convention in Philadelphia,
Pennsylvania (hence the name Legionnaire's Disease). It has a genome of approximately

3.4 mbp in the form of a circular chromosome and also contains plasmids. L. pneumophila
is an acid-fast, non-sporulating, and non-encapsulated rod-like bacterium of approximately
2 µm in length and 0.3-0.9 µm in width, with a single polar flagellum. L. pneumophila is
neither pigmented nor does it autofluoresce.

Transmission is via inhalation of the Legionella pneumophila bacterium, although L.
pneumophila can only be acquired from an environmental source; therefore, infection
never occurs between humans or humans and animals. The bacteria can inhabit amoebae,
being intracellular parasites of free-living protozoa. As such, transmission is often through
contact with contaminated water with protozoa acting as a natural reservoir. Legionella
pneumophila causes a form of pneumonia but does not inhabit the upper respiratory tract
as other bacterial causes of pneumonia do. Rather it enters the alveoli where alveolar
macrophages, and sometimes type II alveolar epithelial cells, phagocytose the L.
pneumophila leading to intracellular growth. The host cell membrane is manipulated to
exclude membrane proteins that trigger the immune system and phagolysosomal fusion is
prevented, allowing the bacteria to persist. Uptake into the endosome allows replication
and expression of virulence factors. Diagnosis of legionellosis can be difficult because
signs and symptoms are non-specific and indistinct from infections caused by other
common causes of pneumonia.

Human infection is caused primarily by a limited number of serogroups, that is serogroups
1, 4, and 6, where serogroup 1 alone is responsible for 70-90% of cases of community­acquired and nosocomial infections. Erythromycin is the antibiotic currently recommended
for treating persons with Legionnaires‘ disease. In severe cases, a second drug, rifampicin,
may be used in addition. Delay in starting appropriate therapy has been associated with
increased mortality.

Introduction to Legionella pneumophila

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MIN

MAX

The L.pneumophila primers have been designed for the specific and exclusive in vitro
quantification of all L.pneumophila isolates and do not detect other Legionella species.
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 pneumophila (L.pneumophila) genomes is
designed for the in vitro quantification of L.pneumophila genomes. The kit is designed to
have the broadest detection profile possible whilst remaining specific to the L.pneumophila
genome.

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

• L.pneumophila specific primer/probe mix (150 reactions BROWN)

FAM labelled

• L.pneumophila 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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