PCRmax Simkania negevensis Instruction Manual

16S ribosomal RNA
(16s rRNA)

Simkania negevensis

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Simkania negevensis (S.negevensis) was formerly referred to as `the microorganism Z' or
`Simkania Z' and belongs to the phylum Chlamydiae. Simkania negevensis is an obligate
intracellular Gram-negative bacterium discovered as a contaminant in a variety of cell
cultures and able to grow in a various free-living amoebae such as Acanthamoeba
poliphaga. Its genome length is 1.7Mbp and the bacterium has been assigned to a new
family Simkanniaceae based on ribosomal DNA sequence comparisons. S . negevensis
differs from members of the Chlamydiaceae in that it is resistant to ampicillin, penicillin G
and cyclosporine. Two alternating developmental forms (elementary bodies and reticulate
bodies) have been observed for this pathogen which is suspected to cause bronchiolitis in
infants, with community acquired pneumonia, and exacerbation of chronic obstructive
pulmonary disease in adults. The elementary bodies are usually smaller, 0.2-0.3 µm in
diameter and the reticulate bodies are typically larger, elongated particles 0.3-0.7 µm in
size.

The bacterium has been shown to be able to replicate in amoeba and survive in amoebal
cycsts in drinking water and in reclaimed waste water. Although contact with infected
secretions or person to person aerosol transmission is possible, it has been shown that
domestic water supplies are also a possible source of infection. The elementary body is
the non-replicating infectious particle that is released when infected cells rupture. The
elementary body is responsible for the bacteria's ability to spread from person to person.
The elementary body contains an RNA polymerase responsible for the transcription of the
DNA genome after entry into the host cell cytoplasm and the initiation of the growth cycle.
Ribosomes and ribosomal subunits are found in these bodies. The elementary body
induces its own endocytosis upon exposure to target cells. Once the host is infected, the
bacterium changes to a reticulate body, which is in fact an intracytoplasmatic form, highly
involved in the process of replication and growth. The reticulate body divides by binary
fission to form particles which, after synthesis of the outer cell wall, develop into new
infectious elementary body progeny. Although the primary target of the pathogen appears
to be the respiratory system, the pathogen has been cultured on epithelial cells from the
gastrointestinal tract and endothelial cells.

There is evidence that human exposure to S. negevensis is already widespread. The
replication cycle of Simkania negevensis, as observed by phase-contrast microscopy,
takes about 12-15 days. Three days after infection by elementary bodies, reticulate bodies
are observed within the cell. These bodies increase in number exponentially within the cell
and 12-15 days following infection, the reticulate bodies may infect surrounding cells.
Aside from bronchitis in infants and pneumonia in adults, S.negevensis has been
suspected of being a cause for lung transplant rejection in adults. Coinfections with other
pathogens have been described, such as respiratory syncytial virus in children and
influenza virus and other bacterial species in adults. S. negevensis DNA has also been
amplified from an aortic aneurysm.

Introduction to Simkania negevensis

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MIN

MAX

Our kit for Simkania negevensis has been designed for the specific and exclusive in vitro
quantification of Simkania negevensis. The 16S ribosomal gene, is the ideal target to
achieve a broad based detection profile for all strains within this species. 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 Simkania negevensis (S.negevensis) genomes is designed for
the in vitro quantification of S.negevensis genomes. The kit is designed to have the
broadest detection profile possible whilst remaining specific to the S.negevensis genome.

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

• S.negevensis specific primer/probe mix (150 reactions BROWN)

FAM labelled

• S.negevensis 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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