PCRmax Neisseria gonorrhoeae Instruction Manual

PorA gene

Neisseria gonorrhoeae

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

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Neisseria gonorrhoeae is a Gram-negative coccus of the Neisseria genus. This bacterium
has a spherical shape with diameter of 0.6-1μm and is usually seen in pairs infecting
human cells. The species has a circular DNA genome of approximately 1Mbp encoding
over 2000 genes. The fimbriae of this bacterium are several micrometers in length and are
essential for host cell adhesion. N. gonorrhoeae is capable of motility via fimbriae
extension, adhesion and contraction.

N. gonorrhoeae is transmitted by sexual contact and usually causes infection in cells of the
mucous membrane of the male urethra or the endocervix and urethra in females. The
bacteria attach to the non-ciliated host columnar, epithelial cells and enter the cell by
endocytosis, mediated by the major porin protein, Por. The endocytosed vacuoles are
transported to the base of the cell where the bacteria are released to replicate at the
basement membrane. During infection, polysaccharides are released from the bacteria that
stimulate host cell production of tumour necrosis factors that cause an inflammatory
response. This inflammation draws neutrophils to the infection site to clear the bacteria by
phagocytosis. However, many bacteria can survive within the phagocyte until they are
released upon neutrophil cell death. One polysaccharide in particular, lipooligosaccharide
(LOS), is also known to cause damage to the mucosal membrane indirectly by promoting
the release of proteases within the host cell.
There is no vaccine against N. gonorrhoeae infection and antibiotic resistance is beginning
to increase. Therefore, treatment is by a course of antibiotics that will be effective against
resistant strains and also treat possible co-infection with Chlamydia.

After a 2-3 day incubation period in males, a purulent discharge from the urethra is
noticeable as well as the development of dysuria. These symptoms can be seen in around
95% of cases of male infection. Women infected with the bacterium are less likely to be
symptomatic, although when symptoms do develop these can be non-specific and can be
confused for other infections; these symptoms can include vaginal discharge, dysuria, and
abdominal pain.
N. gonorhhoeae infections can cause complications when the bacterium enters the blood
or other cells. Complications in males caused by the infection can result in prostatitis or
orchitis if the bacteria spread. In females, invasion of the fallopian tubes or ovaries can
result in salpingitis or ovaritis respectively, with any of these infections possibly resulting in
sterility.

Introduction to Neisseria gonorrhoeae

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MIN

MAX

The primers have 100% homology with all N.gonorrhoeae sequences in the phylogenic
tree below and therefore have the broadest detection profile. The primers have a low
homology to the closely related Neisseria meningitides and will not detect on this strain.

Fig.1 Neisseria Gonorrhoeae PorA Sequences detected

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

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

FAM labelled

• N.gonorrhoeae 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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