year 2, Issue 2 (Summer 2008)
Iran J Med Microbiol 2008, 2(2): 15-25 |
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Shahhosseiny M H, Hosseiny Z, Tabarraii B, Akhlaghi F, Shokrgozar M A, Moslemi E. PCR detection ofMycoplasma spp. contamination in cell culture. Iran J Med Microbiol 2008; 2 (2) :15-25
URL: http://ijmm.ir/article-1-116-en.html
URL: http://ijmm.ir/article-1-116-en.html
Mohhamad Hassan Shahhosseiny1 
, Zahra Hosseiny1 , Bahman Tabarraii2 , Fatemeh Akhlaghi3 , Mohhamad Ali Shokrgozar4 , Elham Moslemi5
, Zahra Hosseiny1 , Bahman Tabarraii2 , Fatemeh Akhlaghi3 , Mohhamad Ali Shokrgozar4 , Elham Moslemi5
1- Department of Microbiology, Islamic azad university, Shahryar unit/Quds branch
2- Department of Microbiology, Islamic azad university, Karaj unit.
3- Razi institute- Quality control Unit
4- Pasteur institute of IRAN-Cell Bank Unit.
5- Department of Biology , Islamic azad university,Eest unit
2- Department of Microbiology, Islamic azad university, Karaj unit.
3- Razi institute- Quality control Unit
4- Pasteur institute of IRAN-Cell Bank Unit.
5- Department of Biology , Islamic azad university,Eest unit
Abstract: (20663 Views)
Background and Objectives: Infections with Mycoplasma species can induce a variety of problems in
living organisms and in in vitro cell cultures. Therefore, it is necessary to establish a routine diagnostic
protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of
commercial biological products. In order to circumvent those limitations, many nucleic acid technology predicated procedures have been developed. PCR-based methods for detection of certain DNA regions of the
Mycoplasma genome have proven both rapid and specific.
Material and Methods: Using SHAH-GPO-3, MGSO primers and standard Mycoplasma species PCR optimized and sensitivity and specificity evaluated by Known CFU samples and different strains. Cell culture DNA extracted and then tested by optimized PCR. Amplicon (272 bp) cloned by PCR-cloning and then sequenced by dideoxy chain termination.
Results: In this study, we describe our newly-developed sensitive PCR procedure for the detection of Mycoplasma genus contaminants. For amplification, the DNA regions of 16S rDNA were targeted using general Mycoplasma primers. The PCR, which generated DNA fragments of 272 bp, was found to be able to detect 10 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. 25 from 47 cell lines infected with Mycoplasmas.
Conclusion:The improved 16S rRNA based PCR method for identification of Mycoplasma in cell culture and biological products, based on common and constant sequences is accurate and useful technique with high specificity and sensitivity. However, more researches should be developed in case of DNA extraction, samples concentration and target sequences and identification of PCR products
Material and Methods: Using SHAH-GPO-3, MGSO primers and standard Mycoplasma species PCR optimized and sensitivity and specificity evaluated by Known CFU samples and different strains. Cell culture DNA extracted and then tested by optimized PCR. Amplicon (272 bp) cloned by PCR-cloning and then sequenced by dideoxy chain termination.
Results: In this study, we describe our newly-developed sensitive PCR procedure for the detection of Mycoplasma genus contaminants. For amplification, the DNA regions of 16S rDNA were targeted using general Mycoplasma primers. The PCR, which generated DNA fragments of 272 bp, was found to be able to detect 10 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. 25 from 47 cell lines infected with Mycoplasmas.
Conclusion:The improved 16S rRNA based PCR method for identification of Mycoplasma in cell culture and biological products, based on common and constant sequences is accurate and useful technique with high specificity and sensitivity. However, more researches should be developed in case of DNA extraction, samples concentration and target sequences and identification of PCR products
Type of Study: Original Research Article |
Subject:
Molecular Microbiology
Received: 2013/11/14 | Accepted: 2013/11/15 | ePublished: 2013/11/15
Received: 2013/11/14 | Accepted: 2013/11/15 | ePublished: 2013/11/15
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