Volume 6, Issue 4 (12-2019)                   nbr 2019, 6(4): 374-381 | Back to browse issues page

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Kabiri K, Majidzadeh K. The molecular detection of the causative agent of plague on the basis of the pla gene. nbr 2019; 6 (4) :374-381
URL: http://nbr.khu.ac.ir/article-1-3084-en.html
The molecular detection of the causative agent of plague on the basis of the pla gene
Khatereh Kabiri * , Keivan Majidzadeh
Department of Microbiology, North Tehran Branch, Islamic Azad University, Tehran, Iran , Kabiri.kh93@ gmail.com
Abstract:   (4352 Views)
Yersinia pestis, a gram-negative rod belonging to the Enterobacteriaceae family, is the causative agent of plague. Classical methods of detecting the organisms are time-consuming, expensive and dangerous. The aim of the study was to design a Real-time PCR assay on the basis of the pla gene of Yersinia pestis. In this research the Real- time PCR test was optimized by using special primers for targeting pla gene. After preparing 10-fold serial dilutions of the pla and their analysis by the assay, the last dilution showing a fluorescent signal was confirmed as the limit of detection (LOD). A standard curve based on the Ct values was depicted, so the assay was developed to quantify the target gene. The analytical specificity was determined by subjecting the genome of some control negative bacteria to the assay. In this experiment, negative control genomes did not show detectable signals in the assay. The last dilution of pla plasmid which showed a fluorescent signal was 4.5 fg. So, the lower detectable copy numbers of the gene in a 20 μl PCR reaction was calculated as 1×103.
 
Keywords: control negative, Real- time PCR, specificity, standard curve, Yersinia pestis
Full-Text [PDF 1176 kb]   (1501 Downloads)    
Type of Study: Original Article | Subject: Microbiology
Received: 2018/02/11 | Revised: 2020/02/24 | Accepted: 2019/05/12 | Published: 2020/01/8 | ePublished: 2020/01/8
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