Hi Kivilcim, I am about a year late, hope my answer were still of use.

Standard material is of capital importance for quantitative viral load assays. You have several sources of virus or DNA samples you can use to control your qPCR assays:

1. You can purchase quantified virus/DNA controls from commercial sources (i.e Advanced Biotechnologies Inc.; Vircell, etc), most advisable, but very expensive.

2. You can obtain your controls from cell lines; the main problem is that you do not know for certain the number of viral genome copies in each cell line, besides that number uses to fluctuate along the culture conditions. To my knowledge, the exception is Namalwa (Burkitt lymphoma cell line) which has 2 EBV integrated copies/genome (be cautious if LMP2a is your target gene) and has been used to prepare standard curves for qPCR "absolute" quantification. Otherwise you can identify productive cell lines, stimulate the lytic cycle (f.i B95.8 for EBV or Dunlop pr BK), centrifuge the virus particles and use them as q standards after careful quantification.

3. You can identify virus-infected cell lines and/or tissues, and then PCR amplify your target sequence, subclone it in a plasmid vector, miniprep it and build your curves by serial log10 dilution. This is the most common way to prepare qPCR standards, but you should be aware of stability and storage issues. Common sources are:

EBV = Namalwa, Raji, B95.8, Burkitt lymphoma tissues (have to screen according to the EBV association frequency in your region). HPV= HeLa (HPV16), CasKi (ATCC CRL-1550, the cells are reported to contain an integrated HPV16 genome at about 600 copies per cell, as well as sequences related to HPV18); cervical tumours. BKV = Dunlop strain (ATCC 45025), urine of transplanted patients (most renal and also bone marrow), have to do some screening.

4. You can ask a commercial supplier (there are several, you have to check in your geographical region) to synthesize your target sequence, you can build all in one plasmid and save time and money.

If you go for (3), you can search in your contry the researchers working with your target virus and ask them to send you a DNA sample. Most researchers will kindly do that, since we all know the difficulties of controlling virus specific PCR!

Thank you very much for your detailed reply, I have just seen it. I still could not find the samples, but I am working on the 3rd option you have presented. Hope it works.