PCRmax Borrelia afzelii Instruction Manual

16S-23S ribosomal RNA
intergenic spacer

Borrelia afzelii

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

1

Borrelia afzelii is a Gram negative aerobic bacterium which is a causative agent of Lyme
disease. It is considered to be the most common tick-borne bacterial disease in the world.
They belong to the spirochete class, genus Borrelia, and have a single stranded linear
DNA genome of approximately 910 kb in length. The genome contains approximately 853
genes coding for basic functions and has several accompanying plasmids. It is a helical
bacterium with an inner and outer membrane as well as a flexible cell wall. The cell is
normally 0.2-0.5µm wide but due to its cylindrical shape can be 5-25µm long. It is
structurally distinct as the flagella are located inside the periplasm between the inner and
outer cell membranes. These allow the cell to travel through the tissue of its infected host
or vector, causing it to be highly invasive.

B.afzelli is primarily transmitted by ticks of the genus Ixodes which serves as both a vector
and a reservoir for the virus. I. scapularis and I. pacificus are the most common vectors
found in the US while I. ricinus and I. persulcatus are found in Europe and Asia. The ticks
usually acquire and transmit the spirochaete form of the bacteria by feeding on an infected
host. The bacteria subsequently reside in the lumen of the tick’s digestive tract,
multiplying whilst remaining there during maturation through the nymph and adult life
cycle stages. The bacteria then migrate to the salivary glands attaching themselves to the
immunosuppressive tick salivary proteins. As an adult, the tick feeds on a new host
resulting in infection of that host at around 24 hours after attachment. The bacterial
infection begins at the site of the tick bite but can spread if untreated.

B. afzelii causes a distinct skin infection known as acrodermatitis chronica atrophicans or
late stage European Lyme borreliosi which is characterised by a progressive fibrosing skin
process. Infection in the skin causes a “bull’s eye” rash around the bite site, sometimes
accompanied by erythema migraine. The rash can be treated with a course of antibiotics
but if left untreated can lead to a disseminated infection through the bloodstream resulting
in neurological problems such as facial palsy or meningitis. A third stage of this infection is
persistent and can be seen several months after the initial infection, causing chronic
symptoms of the heart, brain, nerves and joints including arthritis.

Introduction to Borrelia afzelii

2

MIN

MAX

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

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

3

Kit Contents

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

FAM labelled

• B.afzelii 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

4