Herpes Simplex and Varicella Zoster Viruses

Direct Processing in Neutralization Buffer: An Alternative to MagNA Pure Extraction

December 2009

Based on work with other PCR assays that we perform in our lab, such as those for group A strep and Bordatella pertussis, we had experience using a direct processing method in neutralization buffer that allows us to bypass nucleic acid extraction. With this method, swab samples that arrive in the microbiology laboratory can be placed directly into a conical tube containing neutralization buffer. Then after a brief heating and vortexing step, the sample can then be transferred to the PCR cuvette and undergo amplification. DNA extraction is not necessary.

This would require all samples to arrive as swabs without M5 media, like the swab in the culturette transport tube shown here. Again, if the swab was place in M5 media, then extraction would still be required to remove the phenol red.

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Jump to section:

• Introduction
• The Viruses
• Laboratory Diagnosis
• HSV/VZV Real-time PCR Sample Workflow
• Is Extraction Required for Dermal and Anogenital Specimens?
• Direct Processing in Neutralization Buffer: An Alternative to MagNA Pure Extraction
• Match Up Identifying Information
• Remove Swab from the Culturette Tube
• Heat Swab in Neutralization Buffer
• Sample is Ready for PCR
• A New Workflow for Dermal and Anogenital Swabs
• Direct Processing in Neutralization Buffer: Comparable to DNA Extraction?
• Comparison of Detection - HSV
• Results: Comparison of Amplification Cycle Threshold
• Comparison of Detection - VZV
• Results: Comparison of Amplification Crossing Points
• Questions and Answers
• Questions and Answers
• M5 Media vs Culturette Tubes: Experiment
• M5 Media vs Culturette Tubes: Results
• Conclusions
• Questions?