Biology Reference
In-Depth Information
negative sample under the test conditions. In contrast, the failure to amplify
both IAC and target indicates inhibition.
External Amplification Control (EAC) is an alternative to the IAC. The
EAC is included in a reaction separate to the target test reaction. An EAC
may be identical to the target nucleic acid sequence and thus include both
primer and probe sequences. Thereby, it aids in the identification and evalu-
ation of possible reaction inhibition. 88,92 A successful amplified EAC signals
“less than complete” inhibition in the matrix extract and that absence of
target signal in the test reaction can be considered as a true negative sample
under the given test conditions. In contrast, failure to amplify both EAC
and targets indicates inhibition.
The advantage of an IAC is that the control for inhibition is performed
in the same reaction as the detection of the target sequence. However, if the
IAC concentration is not correctly optimized, this may lead to competi-
tion between amplification of target sequence and IAC. This competition is
not a problem if an EAC is used; however, using an EAC does not control
for some situations such as individual pipetting errors, and using an EAC
increases the number of reactions that are required for each sample. 86
Negative Amplification Control ( Fig. 4.10 ) is a separate amplification reac-
tion in which the nucleic acid extracted from the matrix is replaced with
water (also called a nontemplate control). This control enables one to verify
that the (RT)-PCR mastermix reagents are not contaminated with target
organism, SPC, or its amplicon.
Positive Amplification Control verifies amplification performance of the
target sequence in a reaction, in which a known amount of nucleic acid
from the target organism is included, and the sample nucleic acid is replaced
with water. A target signal must be obtained from the positive amplification
control.
4.6.2. An analytical control in detection of Cryptosporidium
and Giardia analysis
For analysis of Cryptosporidium and Giardia , an internal quality control
consisting of vials containing a known number of (oo)cysts stained red are
commercially available (ColorSeed, BTF, www.btf.com ). The (oo)cysts are
added to the water sample to be filtered or, if the sample has been filtered
elsewhere and the filter sent to the lab or the filter is connected to a tap, they
can be added to the eluate. If so, this will not show how well the filtration
step has worked but only the subsequent concentration step. Staining is
carried out as usual using a positive control (oo(cyst)-suspension) and a
Search WWH ::




Custom Search