Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 99

Sample Process Control—Negative Control is a “sample” in which both
sample matrix and SPC are replaced by sterile distilled water or equivalent.
This control should contain all reagents used in test sample analysis and
verifies that the sample treatment reagents and/or equipment are not con-
taminated with sample process control organism or its amplicon. Neither
target nor SPC must be detected in this sample.
4.6.1.2. Amplification controls
Amplification inhibition is caused by the presence of co-extracted (RT)-
PCR inhibitors. The purity of the nucleic extracts can be estimated
using optical density measurements, and the amplifiability of the prepa-
ration can be tested by (RT)-PCR. The optical density measures the
protein contamination. It is not necessarily correlated to the success of
(RT)-qPCR, 90 as other substances can interfere with this method as well.
Thus (RT)-qPCR provides a better estimate of purity, and even if the
nucleic acid is considered “dirty” by e.g. visual inspection the enzymes
may well be able to amplify the target efficiently. 91 One approach to
assess for—or reduce—inhibition from potential co-extracted inhibitors
is to test for the target organism in undiluted and diluted nucleic acid
extracts. However, this approach also affects the assay sensitivity because
only a smaller amount of the sample is analyzed. Another way to con-
trol for inhibitors present in the nucleic acid preparation is to compare
the amplification efficiency of a target similar but yet distinguishable
template added to the test material extract and a control reaction. 85 The
amplification product from the amplification control could be distin-
guishable from the target template by for example an internal sequence
modified to contain recognition sites for restriction enzymes 91 or an
alternative probe sequence. 75
Amplification controls should be included in the qPCR reagent mix-
ture, or additional reactions performed, depending on their character.
Internal Amplification Control (IAC) is a nucleic acid sequence present in
every reaction. An IAC verifies if amplification reactions have functioned
correctly and identify those which have failed. An IAC is flanked by target
primers to enable simultaneous co-amplification with the target sequence
during (RT)-qPCR detection of the target but is distinguishable from the
target sequence by e.g. an IAC-specific probe. 75 Ideally, an IAC should be
included for both the target organism and the SPC. A successful ampli-
fied IAC 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