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.com
Volume 7, Issue 2 (Suppl)
J Ecosyst Ecography, an open access journal
ISSN:2157-7625
September 18-20, 2017
September 18-20, 2017 Toronto, Canada
Joint Conference
International Conference on
International Conference on
Environmental Microbiology and Microbial Ecology
&
Ecology and Ecosystems
J Ecosyst Ecography 2017, 7:2 (Suppl)
DOI: 10.4172/2157-7625-C1-030
Confirmatory assays for detection of
Neisseria gonorrhoeae
using
PorA
pseudogene real-time PCR base
methods
Imarenezor E P K
Federal University Wukari, Nigeria
Background:
Since the advent of molecular techniques, diagnosis of
Neisseria gonorrhoeae
has been ruin by false positive results
when compared with culture, which is currently the gold standard. False positive results are often due to the cross‐reaction of nucleic
acid amplification test (NAAT) with closely related non-pathogenic
Neisseria
species. Regardless of the availability of commercial
NAATs for
N. gonorrhoeae
, issues surrounding the specificity of these platforms persist.
Objectives:
This research aims to institute, heighten and compare the sensitivity and specificity of previously available
N. gonorrhoeae
real‐time assays which target the
porA
pseudogene.
Methods:
During investigation, 156 gonococci specimens and 30 non-gonococci culture specimens were used. Optimization of
the
PorA
pseudogene real‐time PCR was carried out by varying the concentration of magnesium chloride as follows: 5mM ranges
between 19.08 (4.31) and 23.27 (17.57), 4 mM ranges from 17.18 (1.15) and 22.01 (16.43) and for 3 mM the range is from 21.71
(2.20) and 27.33 (15.27) with the standard deviation in bracket and as well as the forward and reverse primers which has varying
concentration as 50 mM, 300 mM and 900 mM for both.
Results:
The results obtained show the high specificity of the assays for all 156 gonococci culture specimens gave positive results, whilst
the 30 non-gonococci specimens gave negative results. This shows that
PorA
pseudogene real-time PCR is a suitable assay for the
confirmation of putative
N. gonorrhoeae
cultures and can assist in identification, particularly in cases where traditional biochemical
and immunology tests have failed. The potential of the
PorA
pseudogene real‐time PCR to detect the presence of
N. gonorrhoeae
specific DNA directly from clinical samples was then evaluated. An initial experiment was performed which involved the addition
of a primer and probe set which acted as an internal control, it was determined that the internal control did not compromise the
sensitivity of the
PorA
pseudogene real‐time PCR assay and could be used reliably to screen for assay inhibition. The
PorA
pseudogene
real‐time PCR was then used to examine some clinical specimens which had been examined previously at three laboratories, each
of which different commercial
N. gonorrhoeae
NAAT platforms was used. The results from this investigation show a high specificity
evidence of
PorA
pseudogene real-time PCR when compared to previous results obtained from the other laboratories.
Conclusion:
The study has succeeded in establishing to very large extent that the
PorA
pseudogene real‐time PCR is a very valuable
assay for the detection and confirmation of
N. gonorrhoeae
specific DNA from both putative cultures and directly from clinical
samples.
kimarenezor@yahoo.com