Quality Assurance guidelines on molecular biology methods
Please find below some notes on Quality Assurance guidelines for analytical
molecular biology methods that may be used for detection/identification of equine
DNA. Please note that these are for general guidance only and should not be
interpreted as prescriptive rules. In all cases, manufacturer’s instructions, where
available, should be followed. A laboratory’s own Quality Assurance systems must
be followed to ensure that confidence is attributed to any analytical service and that
the application of any analytical method is fit for purpose.
General guidance on molecular biology methods

Positive controls should be included where available to monitor the
performance of the assay and reagents (e.g. authenticated materials, inhouse standards or standards provided with kits being used)

Appropriate negative controls must always be included to monitor
contamination issues and performance of reagents (e.g. PCR no template
controls, extraction blanks, etc.)

Where possible use validated approaches, accredited methods and/or peer
reviewed published methods

Ensure
the
method
being
used
has
published
performance
characteristics/criteria associated with it, particularly: trueness; specificity;
sensitivity (LOD); and measurement uncertainty estimates

Ensure Good Laboratory Practice is always adhered to, to help minimise
chances of DNA contamination

Ensure all equipment is suitable and calibrated

Has the method got proven robustness / commutability / scope appropriate for
the application?

Is a protocol or SOP available?
General guidance on all PCR approaches (real-time PCR and PCR + Capillary
Electrophoresis)
When selecting appropriate assays for PCR, it is preferable to choose those that
have been validated experimentally by a reputable laboratory or study. If assays of
this kind are unavailable, published literature may be consulted for assays that have
been experimentally validated. It is advisable to select assays that are applicable to
the type of PCR that is being carried out (i.e. avoid primers designed for real-time
PCR when carrying out qualitative PCR as these may not be particularly specific,
especially if originally combined with a probe). Pay careful attention to what primer,
master mix and template concentrations were used in the original study, and follow
the recommended cycling conditions.
To ensure that contamination has not occurred during different stages of the
experiment, suitable negative controls should be included. These may include
extraction negative controls (extraction blanks) for which the DNA extraction protocol
is followed in the absence of any sample, and negative template controls (NTCs)
which are produced during PCR setup by replacing template DNA with sterile
molecular grade water. Negative controls should show no PCR amplification.
Amplification of extraction blanks indicates that contamination has occurred during
DNA extraction, and that the extraction batch is invalid. Amplification of NTCs
indicates that contamination has occurred during PCR setup and that the PCR batch
should be repeated. All samples should be prepared again and re-amplified.
Ensure that good laboratory practice is carried out when setting up reactions. This
includes the use of Personal Protective Equipment, cleaning of work surfaces with
an appropriate decontaminant solution, use of fresh oligo/water aliquots where
applicable, correct pipetting practices to ensure accurate volumes are used, and
appropriate disposal of waste material to keep the area clean for future use. Primers
and template DNA should not be used after going through multiple freeze-thaw
cycles, as this could cause degradation and affect amplification. Where possible,
carry out master mix set-up in an area separate to where DNA templates are to be
diluted to avoid contamination with positive controls and/or samples.
Guidance for PCR and Capillary Electrophoresis
Capillary electrophoresis can be carried out on the Agilent Bioanalyzer 2100. All
reagent and chip kits can be purchased from this supplier, which also provide user
manuals specific to each type of chip kit.
Following electrophoresis, the following features can be used as a guide to assess
whether the PCR/CE run has been successful:

With regards to the DNA ladder, 13 peaks should be present for DNA 1000
kits

All peaks should be well resolved, single peaks with a flat baseline

Upper and lower marker peaks should be visible at the correct size

All samples should appear as clear, well resolved peaks between the lower
and upper marker peaks with a flat baseline

Any unused wells (containing water instead of sample) or NTCs should have
no peaks present other than the size markers
Guidance for real-time PCR approaches
Real-time PCR approaches for meat speciation should be sourced with care and
manufacturer’s instructions followed throughout. Care should be taken to ensure that
the approach is fully validated (e.g. performance characteristics including, but not
limited to, specificity, sensitivity, robustness, linearity, range, scope, precision, bias,
measurement uncertainty, LOD and LOQ, should be qualified) and that the
application of the real-time PCR approach is fit for purpose.
Guidance for DNA sequencing
Best measurement practice guidelines should be adhered to. This includes, but is
not limited to: quantification and purification of PCR product; selection of appropriate
sequencing primers; selection of appropriate service provider; provision of
sequencing results; QC reports; choice of database; database alignment; BLAST
similarity results including Query coverage, Max identity and E-values; judgement on
likely candidate sequence(s).