Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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PCR-Based Detection of Mycoplasma Species

  • Sung Hyeran (College of Pharmacy, Chungbuk National University) ;
  • Kang Seung Hye (College of Pharmacy, Chungbuk National University) ;
  • Bae Yoon Jin (College of Pharmacy, Chungbuk National University) ;
  • Hong Jin Tae (College of Pharmacy, Chungbuk National University) ;
  • Chung Youn Bok (College of Pharmacy, Chungbuk National University) ;
  • Lee Chong-Kil (College of Pharmacy, Chungbuk National University) ;
  • Song Sukgil (College of Pharmacy, Chungbuk National University)
  • Published : 2006.02.01
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Abstract

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the generated DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was' achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the disagnosis of mycoplasmal contamination in cell culture systems.

Keywords

References

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