Rapid Detection of Salmonella typhi by Multiplex PCR of invA, tyv, prt genes and sequense comparison of Iranian Isolates with Gene Bank
Trauma Monthly: 11 (1); 1-12 Article Type: Research Article
Z. Rapid Detection of Salmonella typhi by Multiplex PCR of invA, tyv, prt genes and sequense comparison of Iranian Isolates with Gene Bank,
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Aims. Rapid molecular detection of salmonella typhi the causative agent of typhoid. Methods. For rapid and specific detection of salmonella typhus we studied the efficiency of multiplex PCR technology. We used the sets of 6 PCR primers for O, H, and vi antigen genes (invA, tyv,prt) located on chromosomes. Genomic DNA was extracted from standard and clinical isolates and analyzed by different primers. Specificity of the assay was evaluated by different gram- negative and gram- positive. The sensitivity of assay was measured by colony count and DNA concentration. To reduce detection time we performed several optimization reactions and PCR programs. Results. This test was able to differentiate typhoid fever agents from other family of salmonella like: Salmonella thyphimorum, Salmonella paratyphoid A, Salmonella Paratyphoid B, Salmonella paratyphoid C, Salmonella infantis, Salmonella Havana and Salmonella entritideise by combinations of the different size bands produced. Standard and clinical isolates of Salmonella were examined and were accurately identified by this assay. Different gram negative and gram-positive bacteria evaluated specificity of the assay. The sensitivity of the assay revealed that this test was able to detect 1-10 copy number or CFUS of bacteria. By optimization of PCR program with standard thermocycler used in research laboratories (Gradient Ependorf Thermocycler) we were able to shorten the PCR time from 2.5 hrs to 35 minutes in 20 cycles comprising of 7 seconds of denaturation, annealing and extension times. Conclusion. By the methods described here it is possible to rapidly detect and identify Salmonella typhus bacteria within 90 minutes of arrival of specimens in the diagnostic microbiology laboratory. Other interesting findings of this study were the possibility of distinguishing between S. infantis and S. Havana. Sequences from this study are registered in the Gene Bank under accession numbers: AY771363, AY771362 and AY771364 .
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