14th ICID - Poster Abstracts - International Society for Infectious ...

When citing these abstracts please use the following reference:
Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number.
Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1
is available electronically on http://www.sciencedirect.com
Final Abstract Number: 75.027
Session: Diagnostics
Date: Friday, March 12, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
Design of improved polymerase chain reaction (PCR) method containing internal positive
control (IPC) for molecular detection of Yersinia pestis
F. eini 1 , M. fallah raoufi 1 , M. Soleimani 2 , F. azarifard 1 , E. jamshidiyan 1 , K. Majidzadeh 2
1 Tasnim biotechnology research center, tehran, Iran, Islamic Republic of, 2 Tasnim biotechnology
research center, Tehran, Iran, Islamic Republic of
Background: Plague is one of the three epidemic diseases still subject to the international health
regulation and notifiable to the WHO. Yersinia pestis, the causative agent of plague in natural foci
transmitted between rodent and human via wild rodent fleas. So set up of suitable and sure to
surveillance and prevention of the disease is necessary. The molecular methods such as
polymerase chaine reaction (PCR) has allowed improvement of detection methods currently used
in laboratories, although not all of these methods include an Internal Positive Control (IPC) to
monitor for false negative results. Therefore we improved a uniplex and multiplex PCR method
with lower of limit of detection.
Methods: PCR reactions performed with primers which targeted of the caf1 and pla genes
located on the pFra and pPst plasmids and the irp2 chromosomal gene located on the
‘pathogenicity island. For acquired different size of these genes aditional primers were designed.
In each gene two parts required were ligated.The limit of detection determined by performing
PCR reactions on serial dilutions of plasmids containing the coresponding inserts.for evaluating
the specificity, PCR reactions were done for negative control bacteria.
Results: Assays were performed with genome of Y. pestis which produced three DNA fragments
of the expected size 300, 400 and 520 base pairs (bp) corresponding to irp2, caf1 and pla genes
respectively. also, for IPC by the same primers the different fragment of the expected size 150,
500 and 300 base pairs (bp) corresponding to irp2, caf1 and pla genes respectively were
acquired. With The lower limit of detection was 370 copy numbers for caf1 gene and 21 for
pla gene. In PCR reactions for negative control bacteria detectable fragments were not observed.
Conclusion: Our method clearly discriminated Y. pestis DNA bacteria which were tested. The
rapidity, specificity and sensitivity of this procedure with the use of IPC to monitor for false
negative results can make this method suitable for diagnosis and suggest that it can serve as a
useful alternative method for inoculation of laboratory animals or the use of specific culture media
for routine plaque surveillance and outbreak investigations.