Herpes Simplex and Varicella Zoster Viruses
Fast, Efficient, and Accurate Processing for PCR Detection
Well, this appears to be a great process, which could benefit the patient by potentially decreasing turnaround time. The question is whether or not it produces comparable results compared to the method using M5 media and MagNA Pure extraction. In order to determine this, we performed a simple study, where known HSV and VZV PCR positive samples from anogenital and dermal sources that had been previously sent in using a culturette tube were collected in the lab. The culturette transport tubes from these samples still contained the sponges which had surrounded the original swabs during transport, and we know that these sponges retain a significant amount of viral DNA. Therefore, we were able to obtain this DNA by placing two swabs inside of each tube and compressing the sponge around the swabs, thus inoculating the swabs with viral DNA. Obviously, these swabs would not be expected to yield as much viral DNA as the original swab that was sent in, but it provided sufficient viral DNA from a clinical source to allow us to perform a comparison of methods.
To do this, one of these two new swabs was placed in 3mL of M5 media as would normally occur when a swab in a culturette tube is received in the lab. The sample then underwent routine extraction. The other swab was place into a tube containing neutralization buffer (which is abbreviated here as an NB tube). Then, 5 microliters of both the extracted and NB samples were amplified by HSV and VZV PCR on the LightCycler instrument.
Jump to section:
- 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