porcine reproductive and respiratory syndrome

4 th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29 th – July 2 nd , 2003
been tested in our laboratory to prevent false positive results.
As a general practice today in PCR laboratories, samples and
mixes are handled in laminar airflow hoods, which are
regularly decontaminated using ultra-violet (UV-C) light and
bleach, help to avoid false positives. We are running a
recommended laboratory practice for the basic steps of nested
PCR (mix and primer preparation, sample preparation, first
and second PCR) in separate laboratory locations. In addition,
special tube-holders and openers were constructed to
minimise the false positive PCR results (7, 8, 10).
Internal controls to avoid false negative results
It is well documented today that inhibitory effects of
ingredients, like heparin, semen components and other
sample contaminants and/or pipetting errors might lead to
false negative results of the PCR. In such cases the infected
samples tested as negative. To avoid such misleading results,
the use of internal controls (termed "mimics") is
recommended. The mimics are safe indicators of amplification
efficiency. A general rule for mimic construction is to produce
nucleic acid molecules, which are different from the target viral
nucleic acid, both in composition and in size, but having the
same primer-binding sequences. Due to the same primerbinding
nucleotide sequences, template and mimic are coamplified
in the same tube without competition. The size
differences between target and mimic provide an easy way to
discriminate the true product from the mimic (3, 10). An
alternative, particularly when working with RNA viruses, is to
use a second primer set in the reaction, which is specific for
the mRNA of a cellular “housekeeping” gene, which is
constitutively expressed in all cells.
Controls in real-time PCR
When running real-time PCR assays, it is also important to
incorporate internal controls. A practical approach is when a
selected fragment of the host animal genome is co-amplified
as an internal control. By including such an intrinsic control
with its specific reporter fluorophore we obtain information on
the sample quality and on pipetting errors. Simultaneously,
the system shows the amplification of the target nucleotide
sequences and provides safety for the diagnosis.
Validation, standardisation
The work of test validation and standardisation is extremely
important today. Both national and international authorities
require rigorous proof that the diagnostic assays are as
reliable as possible. It is clear today that validation and
standardisation are practical necessities as a “performance
benchmark” if better new and more reliable diagnostic tests
are to be developed and brought into everyday use.
International agencies like the OIE, the Joint FAO/IAEA
Division, national research institutions and commercial
companies make great efforts to agree on international
standardisation (34, 35). Considering these requirements, our
laboratory (together with our partner institutions in Europe)
has started the validation and standardisation of the routine
diagnostic PCR assays.
Diagnostic assay validation
To make predictions about the performance of a diagnostic
method, it is necessary to validate the assay in question.
Validation is the evaluation of the method with the purpose to
determine how fit the assay is for a particular field of use.
General requirements for the competence of testing and
calibration laboratories (EN ISO/IEC 17025:2000)
This standard is from late 1999 and is the standard to be
followed for accredited European laboratories performing
routine diagnostic work and it substituted the standard EN
45001:1989. Basically this standard gives the frame for the
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work of an accredited laboratory and it specifies many
important parameters in such an environment. Part of the
standard is covering issues of validation. It is stated that the
laboratory should validate: non-standardized methods, inhouse
developed methods and standardized methods if
they are used outside the original area of use. The
validation process, routines and results should be
documented and finally a statement by the laboratory
discussing the suitability of the test can be made. The
amount and quality of the work involved in such a validation
process is largely determined by the needs of the
customers. Examples of estimated parameters can be: LoD
(Level of Detection), linearity of the method, reproducibility,
repeatability, robustness or any other parameter interesting
to the customer. This, rather general, standard has been
further developed for the veterinary field by OIE.
The OIE principles of validation
OIE has published (2000; new version is due 2004) a
standard for the validation of diagnostic assays in the
veterinary field. Chapter I.3 in this standard (“Principles of
validation of diagnostic assays for infectious disease”)
describes in detail how to perform validation of the
diagnostic assays in a standardised way. Since this chapter
is of major importance, we provide here shortly the main
points of assay validation according to OIE.
Stage 1. Feasibility studies
The first step in validating a new assay is to perform some
kind of feasibility study. The aim is to determine whether or
not a new assay is suitable to detect a range of virus
concentrations without background activity. Several control
samples (