Fazzeli Hossein, Arabestani Mohammad R, Esfahani Bahram N, Khorvash Farzin, Pourshafie Mohammad R, Moghim Sharareh, Safaei Hajieh G, Faghri Jamshid, Azimian Amir
Department of Microbiology, Isfahan University of Medical Sciences, Isfahan, Iran.
Adv Biomed Res. 2013 Mar 6;2:7. doi: 10.4103/2277-9175.107972. Print 2013.
Throughout the world, bloodstream infections (BSIs) are associated with high rates of morbidity and mortality. Rapid pathogens identification is central significance for the outcome of the patient than culture techniques for microbial identification. To develop an end point multiplex PCR to identify a group of bacteria including Enterococcus spp., Pseudomons aeruginosa, Staphylococcus spp., Acinetobacter baumannii, 16S rDNA, and Drosophila Melanogaster were used as internal control (IC).
Design of primers was done using Mega4, Allel ID6, Oligo6 and Oligo analyzer softwares. Genetic targets for primer designing and identification of genus Enterococcus spp., Staphylococcus spp., and species of Acinetobacter baumannii, Pseudomons aeruginosa, included the rpoB, rpoB and gyrA, sss respectively. Then PCR and multiplex PCR were performed.
The intended specificity was obtained for the bacteria, which used in this study and there wasn't seen any unspecific amplification by the multiplex PCR. The test showed a sensitivity ranging from 1 to 100 target copies per reaction depending on the bacterial species.
The presented multiplex PCR offers a rapid and accurate molecular diagnostic tool for simultaneous detection of some pathogenic microorganisms. The IC exists in the multiplex PCR accompanied by other primers in the system, can serve as a simple, cost- effective internal control for the multiplex PCR assay.
在全球范围内，血流感染（BSIs）与高发病率和死亡率相关。快速鉴定病原体对患者的预后至关重要，其意义超过用于微生物鉴定的培养技术。为开发一种终点多重聚合酶链反应（PCR）以鉴定包括肠球菌属、铜绿假单胞菌、葡萄球菌属、鲍曼不动杆菌在内的一组细菌，16S核糖体DNA（rDNA）和黑腹果蝇被用作内部对照（IC）。
使用Mega4、Allel ID6、Oligo6和Oligo分析仪软件设计引物。用于引物设计以及鉴定肠球菌属、葡萄球菌属、鲍曼不动杆菌种、铜绿假单胞菌的基因靶点分别包括rpoB、rpoB和gyrA、sss。然后进行PCR和多重PCR。
本研究中使用的细菌获得了预期的特异性，多重PCR未出现任何非特异性扩增。根据细菌种类，该检测显示每个反应的灵敏度范围为1至100个靶标拷贝。
所提出的多重PCR为同时检测某些致病微生物提供了一种快速、准确的分子诊断工具。多重PCR系统中与其他引物一起存在的内部对照可作为多重PCR检测简单、经济高效的内部对照。
