We are trying to extract HPV DNA from cervical swabs (ThinPrep) using MagNA Pure 96 equipment from Roche and we are encountering a serie of dificulties.
We have tried several approaches regarding sample preparation:
- 2 ml or 1 ml of cell suspension, wash with PBS, external lysis (1 hour or overnight incubation with Prot. K), take 500 ul directly to the equipament -> 50 or 100 ul of elution
- 500 ul of cell suspension directly to the equipment, without any sample preparation
- 500 ul of cell suspension directly to the equipament, without any sample preparation, and adding 50 ul of PBS 10x
We have used several protocols: Viral Universal, Pathogen Universal, DNA Cells, Viral NA Plasma.
The best results obtained were an agreement rate of 50% with the current extraction: manual protocol using Virus MinElute Kit from QIAGEN. The amplification system is a real time PCR with SYBR Green (Master Mix from Applied Biosystem) or a commercial kit INNO-LIPA from Fujirebio.
Does anyone have a tip to improve this agreement rate to 90 or 100%?
For other types of samples the results are good (plasma, urine, dried swab, LCR) except for the fact that in External Quality Assessment programs the extraction efficiency is very low: the diference in the Ct values are as big as 6 units.
Hi Mario,
It is difficult to answer your question with limited details of your assay. One problem might be the PCR master mix that you are using as it may not be completely compatible with the Roche elution buffer. The Roche PCR master mix might solve this issue for you. I would suggest contacting your local Roche representative for more information. Alternatively, you could contact the UK technical/applications or engineering support team on 0808 100 19 20, (email: [email protected]).
You will find many publications in the literature where various different PCR assays are compared and there are always discrepancies. I think that a kappa value of 0.8 is a good level of agreement. For HPV, it really depends on what your objective is and comparison to HC2 or INNO-LIPA may not give you relevant information.
The Roche HPV assay is run on the cobas 4800 system and is CE-IVD and FDA approved for triage and primary screening. It is approved for use in triage and test-of-cure by the UK NHS-CSP and is extensively clinically validated through the ATHENA trial and other clinical studies conducted world-wide. Please consult the literature (e.g. papers by Wright, Stoler and Huh), the Roche website and www.hpv16and18.co.uk or www.hpv16and18.com
There has recently been a study on vaginal self-sampling conducted in Scotland (PaVDaG study) by Dr Grazyna Stanczuk. A preliminary report has been published and a presentation was made at EUROGIN2015. The full paper is to be published soon and another presentation will be made at IPV in Lisbon this September. If you can come to this conference, please do come to visit the Roche stand where we can discuss your needs and potential solutions.
Best regards,
Nick
How did you determine the 50% value? Was it purely based on PCR, or did you quantify the DNA using some other method?
If you have access to a NanoDrop or other spectrophotometer, you can estimate the quantity and purity of DNA.
A fluorescence based assay, such as Qubit, is good for more accurate quantification of your extract.
Hello Andrew. Thank you for your reply. We quantify the DNA in Nanodrop and there aren't much differences. The 50% value was determined using a real time PCR and comparing both extractions: EZ1 and MagNA Pure.
Regarding Nick's post, thank you for your answer. I'm in contact with the portuguese office from Roche and I will ask their help.
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