PCRmax Batrachochytrium dendrobatidis Instruction Manual

5.8S ribosomal RNA (5.8S)

Batrachochytrium
dendrobatidis

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Batrachochytrium dendrobatidis (B. dendrobatidis) is a fungus responsible for causing
chytridiomycosis in amphibians. The disease has devastated amphibian populations
around the world, in a global decline towards multiple extinctions. The DNA genome is

23.7 Mb in size and it is estimated to consist of approximately 8,800 genes. B.
dendrobatidis exists as a reproductive sporangium and a motile, uniflagellated zoospore
released from the zoosporangium. Zoospores of B. dendrobatidis are typically 3-5 µm in
size, have an elongate–ovoidal body with a single, posterior flagellum 19-20 µm long.

Chytridiomycosis has been linked to dramatic population declines or even extinctions of
amphibian species all over the world and the fungus is responsible for a 100% mortality in
some amphibian populations. Zoospores first encounter amphibian skin and give rise to
sporangia, which produce new zoospores. The disease then progresses as these new
zoospores re-infect the host. The zoospores are capable of chemotaxis, and can move
towards a variety of molecules that are present on the amphibian surface, such as sugars,
proteins and amino acids. B. dendrobatidis also contains a variety of proteolytic enzymes
and esterases that help it digest amphibian cells and use amphibian skin as a nutrient
source. Once the zoospore reaches its host, it forms a cyst underneath the surface of the
skin, and initiates the reproductive portion of its life cycle. The encysted zoospores develop
into zoosporangia, which may produce more zoospores that can reinfect the host, or be
released into the surrounding aquatic environment. The amphibians infected with these
zoospores are shown to die from cardiac arrest.

Morphological changes in amphibians infected with the fungus include a reddening of the
ventral skin, convulsions with extension of hind limbs, accumulations of sloughed skin over
the body, sloughing of the superficial epidermis of the feet and other areas, slight
roughening of the surface with minute skin tags, and occasional small ulcers or
haemorrhage. Behavioural changes can include lethargy, a failure to seek shelter, a failure
to flee, a loss of righting reflex, and abnormal posture (e.g. sitting with the hind legs away
from the body). B. dendrobatidis can grow within temperature range of 4-25°C. The ability
to survive at low temperatures, gives the fungus the ability to overwinter in its hosts, even
in aquatic environments.

Introduction to Batrachochytrium dendrobatidis

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MIN

MAX

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

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

• B.dendrobatidis specific primer/probe mix (150 reactions BROWN)

FAM labelled

• B.dendrobatidis 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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