Nucleic Acid Extraction

Depends on the PCR primers; for a broad range of primers, pure culture of a bacterial/viral agent is generally required for its identification. If PCR primer is designed specifically for a particular microbial agent, clinical specimens may be used directly for nucleic acid extraction. Various DNA extraction methods can be used, such as traditional phenol chloroform method, commercial DNA extraction kits, and so forth. Pure culture and relatively large quantity of target DNA makes contamination by background DNA from reagents and other sources negligible. In our experience, for most bacterial target, no DNA purification is necessary. Two colonies or the pellet of 1 mL positive liquid medium are resuspended in 200 |L sterile saline; 2 |L of the suspension is used directly in the subsequent PCR reaction. Alternatively, the bacterial suspension can be boiled for 10 min and centrifuged for 5 min at 8000 x and the supernatant (2 |L) can be used for PCR.

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