An Improved Method of Elimination of DNA from PCR Reagents

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• 作者：Farjana B. Rowther (1)
  Camilla Rodrigues (2)
  Ajita P. Mehta (2)
  Minal S. Deshmukh (1)
  Farhad N. Kapadia (3)
  Ashit Hegde (3)
  Dr Vinay R. Joshi (4)
  
• 刊名：Molecular Diagnosis & Therapy
• 出版年：2005
• 出版时间：June 2005
• 年：2005
• 卷：9
• 期：2
• 页码：53-57
• 全文大小：205KB
• 参考文献：1. Meier A, Persing D, Finken M, et al. Elimination of contaminating DNA within PCR reagents: implications for a general approach to detection of uncultured pathogens. J Clin Microbiol 1993; 31(3): 646-2
  2. Corless CE, Guiver M, Borrow R, et al. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 2000; 38(5): 1747-2
  3. Ou CY, Moore JL, Schochetman G. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 1991; 10(4): 442-
  4. Klausegger A, Hell M, Berger A, et al. Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria. J Clin Microbiol 1999; 38(5): 1747-2
  5. Hilali F, Saulnier P, Chachaty E, et al. Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes. Mol Biotechnol 1997; 7: 207-6 CrossRef
  6. Carroll N, Adamson P, Okhravi N. Elimination of Bacterial DNA from Taq DNA Polymerase by Restriction Endonuclease Digestion. J Clin Microbiol 1999; 37(10): 3402-
  7. Spielmann P, Dwyer T, Sastry SS, et al. DNA structural reorganization upon conversion of a psoralen furan-side monoadduct to an interstrand cross-link: implication for DNA repair. Proc Natl Acad Sci U S A 1995; 92: 2345- CrossRef
  8. Sastry S, Ross B, P’Arraga A. Cross-linking of DNA binding proteins to DNA with psoralen and psoralen furan-side monoadducts. J Biol Chem 1997 Feb; 272(6): 3715-3 CrossRef
  9. Komura J, Ikehata H, Hosoi Y, et al. Mapping psoralen cross links at the nucleotide level in mammalian cells: suppression of cross-linking at transcription factor or nucleosome-binding sites. Biochemistry 2001; 40(13): 4096-05 CrossRef
  10. Dall’Acqua F, Marciani S, Rodighiero G. Inter-strand cross-linkages occurring in the photoreaction between psoralen and DNA. FEBS Lett 1970; 9(2): 121- CrossRef
  11. Dall’Acqua F, Veldaldi D, Bordin F, et al. New studies on the interaction between 8-methoxypsoralen and DNA in vitro. J Invest Dermatol 1979; 73: 191- CrossRef
  12. Martineau F, Picard F, Roy PH, et al. Species-specific and ubiquitous DNA-based assays for rapid identification of / Staphylococcus epidermidis. J Clin Microbiol 1996; 34(12): 2888-3
  13. Arabzadeh A, Bathaie SZ, Farsam H, et al. Studies on mechanism of 8-methoxyp-soralen-DNA interaction in the dark. Int J Pharm 2002; 237: 47-5 CrossRef
  14. Tseng CP, Cheng JC, Tseng CC, et al. Broad-range ribosomal RNA real-time PCR after removal of DNA from reagents: melting profiles for clinically important bacteria. Clin Chem 2003; 49: 306- CrossRef
  
• 作者单位：Farjana B. Rowther (1)
  Camilla Rodrigues (2)
  Ajita P. Mehta (2)
  Minal S. Deshmukh (1)
  Farhad N. Kapadia (3)
  Ashit Hegde (3)
  Dr Vinay R. Joshi (4)
  
  1. Research laboratories, P.D. Hinduja National Hospital and MRC, Mumbai, India
  2. Department of Microbiology, P.D. Hinduja National Hospital and MRC, Mumbai, India
  3. Department of Intensive Care Medicine, P.D. Hinduja National Hospital and MRC, Mumbai, India
  4. Department of Medicine, P.D. Hinduja National Hospital and MRC, Veer Savarkarmarg, Mahim, Mumbai, 400016, India
  
• ISSN：1179-2000

文摘

Objective: The presence of exogenous DNA in commercially available polymerase chain reaction (PCR) reagent preparations is a serious problem when amplifying conserved regions of bacteria. The preferred and currently in-use method of decontamination using 8-methoxypsoralen (8-MOP) and UVA requires re-standardization of decontamination with increasing concentrations of 8-MOP and UVA irradiation timings, if the DNA load of reagents is high due to lot-to-lot differences. The objective of this study was to develop a decontamination method, which would (i) work at the minimum reported concentration of 8-MOP and UVA irridation timings; and (ii) take care of inter-batch DNA-load variability of reagents. Materials and methods: The improved method described here was formulated after studying the exact molecular mechanism of action of 8-MOP with DNA. The successful working of the method was experimentally proven and validated with 6- new batches of PCR reagents. The sensitivity of eubacterial PCR, after using the new method of decontamination, to be used clinically was checked with both the spiked specimens and the actual clinical specimens. Results and discussion: The new method was found to work at the same starting parameters of 8-MOP and UVA in such situations. The increased efficiency of this method was found to be due to the synergistic effect of both the selective treatment of Taq DNA polymerase and the split-irradiation approach.