M. Fallah Raoufi

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Milad Soleimani

The University of Texas at Austin

Keivan Majidzadeh‐A

Motamed Cancer Institute

F. Azarifard

E. Jamshidiyan

Fatemeh Eini

Hormozgan University of Medical Sciences

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University of Tehran

The University of Texas at Austin

Livestrong Foundation

The University of Texas at Arlington

American University of Ras Al Khaimah

Tarbiat Modares University

Motamed Cancer Institute

Academic Center for Education, Culture and Research

Cancer Research Center

Aja University of Medical Sciences

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Design of improved polymerase chain reaction (PCR) method containing internal positive control (IPC) for molecular detection of Yersinia pestis

International Journal of Infectious Diseases (2010)

Fatemeh Eini M. Fallah Raoufi Milad Soleimani F. Azarifard E. Jamshidiyan

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. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

Yersinia pestis

Primer (cosmetics)

plague

10.1016/j.ijid.2010.02.427

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Development of a multiplex PCR system with application of IPC (internal positive control) for detecting Bacillus anthracis from environmental samples

International Journal of Infectious Diseases (2010)

M. Fallah Raoufi Fatemeh Eini F. Azarifard Milad Soleimani E. Jamshidiyan

Background: Bacillus anthracis, the causative agent of anthrax is a major concern of a dangerous and zoonotic disease in medicine and veterinary medicine. The development of rapid, sensitive and simple techniques to detect B.anthracis in suspicious specimens is the important aim of animals and subsequently public health. we described a multiplex PCR system with application of IPC (internal positive control) for detecting virulence markers in B.anthracis. Methods: In PCR assay pag and cap genes located on POX1 and POX2 plasmids were amplified by use of specific primers. In order to control of reaction conditions, we used from PCR products as a templet and designed IPC for pag and cap genes that their length was shorter than product segments. After cloning of PCR products, the sensitivity of the PCR test (limit of detection) was also estimated. Control bacteria of defined species was confirmed by amplification of a fragment of the 16S rRNA gene by using two universal primers. Results: In post-PCR stage 591 and 173 bp fragments of DNA respectively to cap and pag genes recognized in agarose gel. Sensitivity determination assays revealed that this method can detect, 412 and 100 copies as LOD for cap and pag gene respectively in samples.Nonetheless, bands on the results with negative controls, these genes not present in these defined bacteria. Conclusion: In this study, we desigined and optimized a multiplex PCR assay for the detection and characterization of B.anthracis from environmental samples. In addition to we used from IPC to prevention of false negative results and also to detect inhibitory effects of the sample matrix. The PCR system described here can be proposed as rapid, safe, diagnostic and confident method for detecting anthrax. The sensitive and specific nature of this assay provides a valuable tool that can be used for analysing clinical and field samples and for improving our understanding of the ecology and environmental prevalence of B.anthracis in natural foci of disease. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

Bacillus anthracis

Multiplex

10.1016/j.ijid.2010.02.428

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