PCRmax Bordetella pertussis Instruction Manual

Insertion sequence IS481/Toxin
subunit 1 precursor (ptxA) gene

Bordetella
pertussis_IS481/ptxA

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Bordetella pertussis is a Gram-negative bacterium of the genus Bordetella, and the
causative agent of pertussis or whooping cough. Unlike B. bronchiseptica, B. pertussis is
non-motile. There does not appear to be a zoonotic reservoir for B. pertussis, humans are
its only host. The bacterium is spread by coughing and by nasal drops. The incubation
period is 7-14 days Whooping Cough, or Pertussis, is an infection of the respiratory system
and characterized by a “whooping” sound when the person breathes in. In the US it killed
5,000 to 10,000 people per year before a vaccine was available. Vaccination has
transformed this and between 1985-88 less than 100 children died from pertussis.
Worldwide in 2000, according to the WHO, around 39 million people were infected
annually and about 297,000 died.

Whooping Cough occurs most with children under the age of one when they are
immunized or children with faded immunity, normally around the age 11 through 18. The
signs and symptoms are similar to a common cold: runny nose, sneezing, mild cough, and
low-grade fever. After a spell, they might make a “whooping” sound when breathing in or
vomit. Adults have milder symptoms, like prolonged coughing without the “whoop.”
Pertussis is highly contagious and may become airborne when the person coughs,
sneezes, or laughs. People infected by this disease are more contagious in the earliest
stages of it, normally 2 weeks after the coughing begins. Whooping Cough can be
prevented by the Pertussis Vaccine which is part of the DTaP (diphtheria, tetanus, acellular
Pertussis) immunization. The paroxysmal cough precedes a crowing inspiratory sound
characteristic of pertussis. (Infants less than 6 months may not have the typical whoop.) A
coughing spell may last a minute or more, producing cyanosis, apnoea and seizures. A
prolonged cough may be irritating and sometimes a disabling cough may go undiagnosed
in adults for many months.

Bodetella pertussis also produces a lymphocytosis-promoting factor, which causes a
decrease in the entry of lymphocytes into lymph nodes. This can lead to a condition known
as lymphocytosis, with a complete lymphocyte count over of 4000/μL in adults or over
8000/μL in children.

The Insertion sequence 481 (IS481) is a high copy number target that is present in the B.
pertussis genome at 80 to 100 copies. The toxin subunit 1 precursor (ptxA) gene is a
single copy number target that will detect the pertussis toxin that is produced by Bordetella
pertussis, this toxin plays a central role in the pathogenesis of pertussis.

Introduction to Bordetella pertussis_IS481/ptxA

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MIN

MAX

The PCR Max qPCR Kit for Bordetella pertussis_IS481/ptxA (B.pertussis-IS481/ptxA)
genomes is designed for the in vitro quantification of B.pertussis-IS481/ptxA genomes.
The kit is designed to have the broadest detection profile possible whilst remaining specific
to the B.pertussis-IS481/ptxA genome.

The primers and probe sequences in this kit have 100% homology with a broad range of
B.pertussis-IS481/ptxA 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.pertussis-IS481/ptxA specific primer/probe mix (150 reactions BROWN)

FAM labelled

• B.pertussis-IS481/ptxA 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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