PCRmax Neospora caninum Instruction Manual

Nc5 marker genomic sequence

Neospora caninum

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Neospora caninum is a protozoan, coccidian parasite of the Sarcocystidae family. It
primarily infects dogs and cattle but is also associated with sheep, goats, deer and horses.
It is the causative agent of neosporosis world wide and shows strong similarity to T.gondii,
although it is antigenically distinct. It has a DNA genome of approximately 3.5 Mb and the
tachyzoites are 2-6 μM in length with a pointed anterior end and rounded posterior end.

Transmission occurs in a cycle between dogs (the definitive host) and mainly cattle
(intermediate host). Dogs pass the N. caninum oocysts in their faeces which are ingested
by cattle grazing on land on which dogs have had contact. Ingestion of infected placental
tissue or infected carcasses by dogs continues the cycle. Transplacental transmission also
occurs in dogs and sheep which can lead to abortion, premature birth or birth of an animal
with a persistent infection. The life cycle of N. caninum is not well understood but it cycles
through tachyzoite, bradyzoite, oocyst, sporocyst and sporozoite stages. The rapidly
replicating tachyzoite stage has been described in many tissue types as the disease
spreads, although latent stage bradyzoites have only been observed in thick-walled tissue
cysts in the central nervous system and the eye. Stimulation of the host immune system
leads to transformation of the tachyzoites to bradyzoites which can persist in the tissue
cysts.

Neospora caninum is an important cause of spontaneous abortion and infertility in cattle,
with a reduction in milk production also observed in adult dairy cows, possibly as a result of
previous abortion. In a non-pregnant animal, infection results in seroconversion and
persistent infection. It may lead to abortion, premature birth or birth of brain damaged or
weak calves depending on the stage of gestation at which the calf becomes infected. Dogs
show signs of rigid hyperextension, forelimb ataxia, head tremors, myocarditis and
pneumonia. The disease can prove fatal in young puppies often with several litter mates
being affected. It can cause neurological signs, especially in congenitally infected puppies,
where it can form cysts in the central nervous system.

Introduction to Neospora caninum

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MIN

MAX

Our kit for Neospora caninum has been designed for the specific and exclusive in vitro
quantification of the Neospora caninum genome. The Nc5 marker genomic sequence
has previously been shown to be a good marker for the detection of Neospora caninum
(Collantes-Fernández E. et. al., 2002).

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 Neospora caninum (N.caninum) genomes is designed for the
in vitro quantification of N.caninum genomes. The kit is designed to have the broadest
detection profile possible whilst remaining specific to the N.caninum genome.

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

• N.caninum specific primer/probe mix (150 reactions BROWN)

FAM labelled

• N.caninum 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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