Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia
Background and Objectives: Streptococcus pneumoniae accounts for bacterial meningitis and is an important cause of morbidity among children and elderly. Control of this disease depends on rapid detection of the causative bacteria. The methods for detection of Streptococcus pneumoniae are gram staini...
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| Format: | Article |
| Language: | fas |
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Qom University of Medical Sciences
2013-09-01
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| Series: | Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Qum |
| Subjects: | |
| Online Access: | http://journal.muq.ac.ir/index.php/qumsj/article/view/763 |
| _version_ | 1811284443334180864 |
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| author | Amir Hossein Mohseni Mohammad Soleimani Fahimeh Aghamiri Keivan Majidzade |
| author_facet | Amir Hossein Mohseni Mohammad Soleimani Fahimeh Aghamiri Keivan Majidzade |
| author_sort | Amir Hossein Mohseni |
| collection | DOAJ |
| description | Background and Objectives: Streptococcus pneumoniae accounts for bacterial meningitis and is an important cause of morbidity among children and elderly. Control of this disease depends on rapid detection of the causative bacteria. The methods for detection of Streptococcus pneumoniae are gram staining, culture, and serological tests. These tests are time consuming and are limited by antimicrobial agents leading to false negative results. Currently, molecular methods such as PCR are used routinely for detection of infectious organisms. This study was performed with the aim of designing an improved PCR assay for the detection of Streptococcus pneumoniae.Methods: The specific diagnostic primers were designed based on ply gene of the bacterium. After amplifying the target gene on the genomic DNA, the PCR product was cloned in pTZ57R/T plasmid and the confirmed pTZ-ply plasmid was used as positive control in next experiments. Sensitivity of the assay was determined by performing the PCR on 10-fold serial dilutions of pTZ-ply. Specificity of the assay was determined using the genomic DNA of other related or unrelated bacterial species.Results: The PCR, as expected, generated a 727bp amplicon. No PCR amplification was observed on the genome of negative controls. These findings indicate high specificity of the PCR. The lowest limit of detection of the assay in the detection of the ply gene was 250 copies in a 25µl reaction.Conclusion: The high sensitivity, specificity, and rapidity of the designed assay suggested the assay as an appropriate test for use in clinical laboratories. The further evaluation of the assay using clinical samples or artificially contaminated materials will confirm the application of this assay in clinical settings. |
| first_indexed | 2024-04-13T02:29:30Z |
| format | Article |
| id | doaj.art-a214e20ee5d1467cb9f62d8e3cd9ca33 |
| institution | Directory Open Access Journal |
| issn | 1735-7799 2008-1375 |
| language | fas |
| last_indexed | 2024-04-13T02:29:30Z |
| publishDate | 2013-09-01 |
| publisher | Qom University of Medical Sciences |
| record_format | Article |
| series | Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Qum |
| spelling | doaj.art-a214e20ee5d1467cb9f62d8e3cd9ca332022-12-22T03:06:39ZfasQom University of Medical SciencesMajallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Qum1735-77992008-13752013-09-01748188Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumoniaAmir Hossein MohseniMohammad SoleimaniFahimeh AghamiriKeivan MajidzadeBackground and Objectives: Streptococcus pneumoniae accounts for bacterial meningitis and is an important cause of morbidity among children and elderly. Control of this disease depends on rapid detection of the causative bacteria. The methods for detection of Streptococcus pneumoniae are gram staining, culture, and serological tests. These tests are time consuming and are limited by antimicrobial agents leading to false negative results. Currently, molecular methods such as PCR are used routinely for detection of infectious organisms. This study was performed with the aim of designing an improved PCR assay for the detection of Streptococcus pneumoniae.Methods: The specific diagnostic primers were designed based on ply gene of the bacterium. After amplifying the target gene on the genomic DNA, the PCR product was cloned in pTZ57R/T plasmid and the confirmed pTZ-ply plasmid was used as positive control in next experiments. Sensitivity of the assay was determined by performing the PCR on 10-fold serial dilutions of pTZ-ply. Specificity of the assay was determined using the genomic DNA of other related or unrelated bacterial species.Results: The PCR, as expected, generated a 727bp amplicon. No PCR amplification was observed on the genome of negative controls. These findings indicate high specificity of the PCR. The lowest limit of detection of the assay in the detection of the ply gene was 250 copies in a 25µl reaction.Conclusion: The high sensitivity, specificity, and rapidity of the designed assay suggested the assay as an appropriate test for use in clinical laboratories. The further evaluation of the assay using clinical samples or artificially contaminated materials will confirm the application of this assay in clinical settings.http://journal.muq.ac.ir/index.php/qumsj/article/view/763Polymerase Chain ReactionPolymerase Chain Reaction-MethodsRapid DetectionStreptococcus pneumoniae. |
| spellingShingle | Amir Hossein Mohseni Mohammad Soleimani Fahimeh Aghamiri Keivan Majidzade Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Qum Polymerase Chain Reaction Polymerase Chain Reaction-Methods Rapid Detection Streptococcus pneumoniae. |
| title | Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia |
| title_full | Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia |
| title_fullStr | Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia |
| title_full_unstemmed | Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia |
| title_short | Design of an Improved Polymerase Chain Reaction (PCR) Assay for Molecular Detection of Streptococcus pneumonia |
| title_sort | design of an improved polymerase chain reaction pcr assay for molecular detection of streptococcus pneumonia |
| topic | Polymerase Chain Reaction Polymerase Chain Reaction-Methods Rapid Detection Streptococcus pneumoniae. |
| url | http://journal.muq.ac.ir/index.php/qumsj/article/view/763 |
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