PCRmax Mycobacterium marinum and M. ulcerans Instruction Manual

Polyphosphate kinase (ppk)
gene, partial cds

Mycobacterium
marinum &
Mycobacterium
ulcerans

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Mycobacterium ulcerans is a human pathogen responsible for Buruli ulcer, a necrotizing
skin disease most commonly found in West Africa, but outbreaks have also been reported
in the Americas, Australia, and Asia.

Mycobacterium marinum (formerly M. balnei) is a free-living bacterium, which causes
opportunistic infections in humans. M. marinum sometimes causes a rare disease known
as aquarium granuloma, which typically affects individuals who work with fish or keep
home aquariums.

M. Ulcerans’ genome consists of approx. 5.8Mbp which codes for around 4240 proteins.
The accepted standard for molecular identification of mycobacteria is sequencing analysis
of 2 hypervariable regions identified in 16S rRNA gene. M. marinum and M. ulcerans share
identical 5´-16S rDNA and 16S-23S rRNA gene spacer sequences Real-Time Polymerase
chain reaction (qPCR) methods are based on the 16S rRNA gene, the hsp65 gene or the
insertion sequence IS2404

After inoculation into the skin, M. ulcerans proliferates extracellularly and elaborates a
toxin, mycolactone, that enters the cells and causes necrosis of the dermis, panniculus,
and deep fascia. Early lesions are closed, but as the necrosis spreads, the overlying
dermis and epidermis eventually ulcerates. Clumps of extracellular acid-fast bacilli are
plentiful and are frequently limited to the base of the ulcer and adjacent necrotic
subcutaneous tissue. With healing, there is a granulomatous response, and the ulcerated
area is eventually replaced by a depressed scar.
Lesions are usually single and begin as firm, painless, non-tender, movable, subcutaneous
nodules 1 to 2 cm in diameter or as small papules. In 1 or 2 months, the nodule may
become fluctuant and ulcerates, with an undermined edge that often extends 15 cm or
more. The skin adjacent to the lesion, and often that of the entire corresponding limb, may
be swollen by edema.

The inhibition of growth of M. marinum at 37°C is related to its ability to infect the cooler
parts of the body especially the extremities. Lesions appear after an incubation period of
about 2–4 weeks, and after 3–5 weeks they are typically 1-2.5 cm in diameter.

Diagnosis is frequently delayed, probably due to the rarity of the infection and a failure to
elicit the usual history of aquatic exposure. Common misdiagnoses include fungal and
parasitic infection, cellulitis, skin tuberculosis, rheumatoid arthritis, foreign body reaction,
and skin tumor. A high index of suspicion and a detailed history are important in
establishing the diagnosis of Mycobacterium infection. Long delays in diagnosis can result
in severe, destructive infection. Sometimes, cultures are negative but the diagnosis is still
made based on physical signs supported by typical histological findings.

The management of Mycobacterium infections depends on the severity of the infection. A
prolonged course of antibiotic therapy is curative in most superficial cases but adjunctive
surgical intervention is sometimes indicated in extensive and deep infections.

Introduction to Mycobacterium marinum &
Mycobacterium ulcerans

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MIN

MAX

The PCR Max qPCR Kit for Mycobacterium marinum & Mycobacterium ulcerans (M.
marinum/M.ulcerans) genomes is designed for the in vitro quantification of M.marinum/M.
ulcerans genomes. The kit is designed to have the broadest detection profile possible
whilst remaining specific to the M.marinum/M.ulcerans genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
M.marinum/M.ulcerans 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

• M.marinum/M.ulcerans specific primer/probe mix (150 reactions BROWN)

FAM labelled

• M.marinum/M.ulcerans 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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