PCRmax African trypanosomiasis Instruction Manual

18S ribosomal RNA (18S) gene

African
Trypanosomiasis

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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African trypanosomiasis also known as sleeping sickness, African lethargy or Congo
trypanosomiasis. It is a parasitic disease of humans and animals caused by Trypanosoma
brucie gambiense in Western Africa and Trypanosoma brucie rhodensiense in Eastern
Africa. Both protozoan species are morphologically indistinguishable, measuring 25-40µm
in length, but have drastically different epidemiological features. Trypanosomes have a
two-unit genome, a nuclear and a mitochondrial (kinetoplast) genome with the entire DNA
of the mitochondrion localized in the kinetoplast.

Several species of hematophagous Glossina, commonly known as the tsetse flies, act as
vectors for the disease and are responsible for the cyclical transmission of the parasitic
protozoan between numerous vertebrate hosts. The tsetse fly becomes infected by feeding
from an infected host. In the midgut of the insect vector, the trypanosomes develop into
procyclic mastigotes eventually migrating into the salivary glands. Infection begins when
the parasite is injected intradermally by the tsetse fly into a mammalian host at its infective
stage known as the metacyclic stage. The organism enters the bloodstream through the
lymphatic system and divides rapidly by binary fission. The trypanosomes finally enter the
central nervous system with serious pathological consequences for the host.

During the first stage of infection the parasite is found in the peripheral circulation, and in
the second stage the parasite crosses the blood brain barrier and infects the central
nervous system. T.b.rhodesiense (East African Sleeping sickness) infection progresses
rapidly. Within one to weeks the patient starts developing fever, body aches and enlarged
lymph nodes. Within months the parasite invades the central nervous system causing
mental deterioration and eventually death. In contrast, the T.b.gambiense infection (West
African Sleeping sickness) progresses very slowly. It takes about one to two years to show
any evidence that there has been an effect on the nervous system. The patient exhibits
neurological signs, such as partial paralysis, hormonal imbalances and the disease more
often causes death in around 3 years.

Introduction to African Trypanosomiasis

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MIN

MAX

Our kit for African Trypanosomiasis has been designed for the specific and exclusive in
vitro quantification of all T.brucei sub species. The 18S ribosomal gene, is the ideal
target to achieve a broad based detection profile for all subspecies including T. brucei: T.
b. brucei, T. b. gambiense and T. b. rhodesiense . 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 African Trypanosomiasis (A.trypanosomiasis) genomes is
designed for the in vitro quantification of A.trypanosomiasis genomes. The kit is designed
to have the broadest detection profile possible whilst remaining specific to the A.
trypanosomiasis genome.

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

• A.trypanosomiasis specific primer/probe mix (150 reactions BROWN)

FAM labelled

• A.trypanosomiasis 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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