PCRmax Burkholderia mallei Instruction Manual

VirG and VirA genes

Burkholderia mallei

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Burkholderia mallei is a rod-shaped, Gram negative coccobacillus of the Burkholderiaceae
family. It is the causative agent for glanders, also known as equinia. Although most known
members of the Burkholderiaceae family are commonly found in the soil, B. mallei is an
obligate mammalian pathogen. Horses are its primary host but mules, goats, dogs and
cats are also susceptible to infection. The disease is endemic in Africa, Asia, the Middle
East and South America. The genome consists of two circular chromosomes comprising of
approximately 5.8 mb. Chromosome 1 contains 3.5 mb and chromosome 2 contains 2.3
mb. A total of 5,535 predicted protein-encoding ORFs have been identified in the genome.
Its sequence is riddled with insertion sequences that have had created a unique effect on
its chromosomal structure.

The primary route of equine infection is most likely the consumption of feed or water
contaminated with nasal discharges of infected animals, although a cutaneous form also
exists, known as farcy. B. mallei infects and gains access to the cell of its host through
lysis of the entry vacuole and has bacterial protein dependent actin-based motility once
inside the cell. It is also able to initiate host cell fusion that results in multi-nucleated giant
cells (MNGCs). The consequence of MNGCs is not well understood, but it may allow the
bacterial spread, persistence or host evasion. B. mallei is able to survive inside host cells
through its capabilities in disrupting the bacteria killing functions of the cell. It leaves the
vacuoles early, which allows for efficient replication of the bacteria inside the cell. Leaving
the cell early also keeps the bacteria from being destroyed by lysosomal defensins and
other pathogen killing agents.

Chronically infected animals present a variety of signs and symptoms dependent on the
route of infection including mucopurulent nasal discharge, lung lesions and nodules
involving the liver and spleen. Acute infection results in high fever and emaciation, with
ulceration of the nasal septum, accompanied by mucopurulent to hemorrhagic discharge.
Inhalation of aerosol or dust containing the bacteria can lead to septicemic, pulmonary, or
chronic infections of the muscle, liver and spleen. The disease has a 95% fatality rate for
untreated septicemic infections and a 50% fatality rate when standard antibiotic treatments
are administered. The course of infection depends upon the nature of exposure. Direct
contact with a cut or scratch can lead to a localized cutaneous infection and ulceration.
Equines that have recovered from glanders develop no protective immunity and thus are
susceptible to future infections.

Introduction to Burkholderia mallei

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MIN

MAX

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

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

FAM labelled

• B.mallei 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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