蓝海人类学在线 Ryan WEI's Forum of Anthropology

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发表于 2014-11-27 16:26 | 显示全部楼层 |阅读模式

The method used for the Taqman assay was as follows: A 20× assay mix
was made using forward primer 5′-GCA ATT CCG ATA GTA ATG GTC A-3′,
reverse primer 5′- CTT GTT TGG CCT TTC ACA AA-3′, and probe /56-FAM/AGTAA ACC CAC ACC CTT TGG TAG CCA /36-TAMSp/ containing 18-μM primers
and a 4-μM probe. A 20-μL reaction volume was used using 10 μL of 2× Gene
Expression Master Mix (Applied Biosystems) and 1 μL of the 20× assay mix
with the remaining volume consisting of input DNA and sterile water. The
qPCR reaction profile consisted of one cycle of 10 min at 95 °C followed by
45 cycles of 15 s at 95 °C and 1 min at 60 °C. Reactions were performed in
a Stratagene Mx3005P Thermocycler (Agilent Technology). Results were
quantified with a standard curve generated from the testing of 10-fold
dilutions of a plasmid created to contain the target. Testing of this standard
curve indicated that 10 copies of ATCV-1 DNA could be reliably detected in
the qPCR reaction. A sample, which contained ≥10 copies of ATCV-1 genomic
DNA, was considered to contain ATCV-1 DNA. DNA extracted from related
chloroviruses PBCV-1 and CVM-1 (13, 29) and human DNA did not
produce products with this assay.


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