Download Comparison of a reverse transcription-polymerase chain

Rev. sci. tech. Off. int. Epiz., 1998,17 (3), 674-681
Comparison of a reverse transcription-polymerase
chain reaction assay and virus isolation for the
detection of classical s w i n e fever virus
S. V y d e l i n g u m T . T a o , K. Balazsi & R. Hecker
(2)
(2)
(1) Pasteur Mérieux Connaught, Bld 8 1 , Room 307A, 1755 Steeles Avenue West, North York, Ontario M2R 3T4,
Canada
(2) Animal Diseases Research Institute, 3851 Fallowfield Road, P.O. Box 11300, Station H, Nepean,
Ontario K2H 8H9, Canada
Summary
The authors evaluated the ability of a reverse transcription-polymerase chain
reaction (RT-PCR) assay to detect classical swine fever virus (CSFV) in
comparison with virus isolation and detection by an indirect immunoperoxidase
assay (VI-IPA).To determine the specificity of the assay, samples from 60 spleens,
45 tonsils, ten submandibular lymph nodes, eight mesenteric lymph nodes and
four kidneys, collected from pigs of various ages which had been slaughtered in
abattoirs in Canada (a population free from CSFV), were tested. All the samples
tested gave negative results by both VI-IPA and RT-PCR. A total of 20 samples
were passaged in porcine kidney (PK) 15 cells and retested by both assays. All
were found to be negative, giving a specificity of 100%.
To determine the analytical sensitivity of the assay, a similar comparative study
was conducted, using CSFV grown in tissue culture and tonsil tissues from a
CSFV-infected pig. For both infected tissues and tissue culture fluids, RT-PCR was
ten times more sensitive than VI-IPA. Amounts as small as 0.6 infectious units per
100 mg of tissue were detected by RT-PCR, compared to 6 infectious units by
VI-IPA. Similarly, RT-PCR could detect as little as 0.1 infectious unit per ml in tissue
culture fluids, compared to one infectious unit per ml by VI-IPA.
To determine diagnostic sensitivity, three coded panels (two internal and one
external), comprising 45 samples from 14 pigs, were tested. The diagnostic
sensitivity of both RT-PCR and VI-IPA was found to be 100% for both internal
panels. The results of the external panel, apart from two samples that were
missed by both RT-PCR and VI-IPA, were found to be in total agreement. These
two samples remained negative after amplification in PK15 cells. All the RT-PCR
results were based on a single test whereas, for the VI-IPA results, positive
results were obtained for five samples only after an amplification round in PK15
cells. Application of the RT-PCR assay for the diagnosis of CSFV would enable
improved detection of the virus in a shorter time period.
Keywords
Classical swine fever virus - Detection - Indirect immunoperoxidase assay - Reverse
transcription-polymerase chain reaction.
Introduction
Classical swine fever (CSF) (hog cholera) virus is an
enveloped ribonucleic acid (RNA) virus which infects pigs.
CSF virus (CSFV), together with Border disease virus and
bovine viral diarrhoea virus, constitute the Pestivirus genus
within the Flaviviridae family (19). The virus has an RNA
genome of about 13,000 kilobases, which codes for
approximately 4 , 0 0 0 amino acids. It is a non-polyadenylated
RNA which is single-stranded and positively polarised (9).
675
Rev. sci. tech. Off. int. Epiz., 17 (3)
The virus is responsible for a devastating disease of pigs,
known as classical swine fever, which has inflicted major
economic losses on the pig industry (8, 10, 16). The disease
may run an acute, subacute, chronic or clinically nonapparent course. Mortality among the pig populations
infected with the virus ranges from 1 0 0 % in acute cases to
none in non-apparent infections. The outcome of the disease
depends on the CSFV strain involved as well as on the
immune status, age and nutritional status of the animal (3).
Classical swine fever is a notifiable disease, which does not
exist in many parts of the world, such as Canada (3). In
countries where the disease does not exist, measures are taken
to prevent its introduction. If, in spite of those measures, the
disease is introduced through infected pigs or contaminated
pig products, steps are taken to ensure that the infection is
contained and eradicated. To ensure prompt and effective
intervention if the disease is introduced, or to prevent the
introduction of the disease itself, virus detection is essential.
Clinical signs and lesions, used historically to monitor the
disease, are no longer considered sufficient. Detection of
antigens (1), antibodies (17) and isolation of the virus (12) are
the most reliable methods employed to date. The detection of
the CSFV genome has also been made possible with the
advent of the polymerase chain reaction (PCR) technique for
testing ( 1 3 , 1 4 ) . Owing to its sensitivity, specificity and speed,
PCR offers definite advantages over conventional methods of
detection. A reverse transcription-PCR (RT-PCR) for the
detection of CSFV was developed in the laboratory of the
authors (5) and has been partially evaluated for use as a
diagnostic test (6). This report describes a systematic
evaluation of the assay and assesses its use as a routine
diagnostic test as compared to virus isolation. Development of
such an assay could be a potentially significant addition to the
repertoire of tests already available for the diagnosis of CSFV.
Materials and methods
Tissue samples
Samples were collected from pigs at abattoirs in Quebec,
Canada (a population free from CSFV). Tonsils were
harvested from pigs of different ages, as follows:
-
20 from market hogs
19 from adult pigs
4 from two-month-old pigs
2 from three-week-old pigs.
with the Standard strain at eight to eleven days post-infection,
and from a piglet infected with the United Kingdom (UK)
isolate at 2 3 days post-infection (6, 2 0 ) , and stored at - 7 0 ° C
until use. Pigs were infected with BAI Nervous or New South
Wales strains of CSFV and euthanased at eight and eleven
days post-infection, respectively, then left at room
temperature for six hours to mimic field conditions. Tissues
were removed and left at 4°C for 4 8 hours to simulate
transportation to the laboratory, before being stored at —70°C.
Preparation of panels and sample processing
Three coded panels were evaluated. Two of these panefs,
consisting of 2 5 samples in total, were set up in the laboratory
of the authors and the third, containing 20 samples from pigs
naturally infected with CSFV, was provided by the National
Veterinary Services Laboratories in Ames, Iowa, United States
of America. Tissue samples were weighed, minced and
homogenised in Dulbecco's minimum essential medium
(DMEM) to produce a 1 0 % (weight/volume) emulsion.
Nucleic acid isolation
Total RNA was isolated as previously described (6). In brief,
samples were digested with proteinase K at 37°C for two
hours for tissue culture samples and at 56°C overnight for
tissue. This was followed by phenol-chloroform-isoamyl
alcohol extraction and ethanol precipitation at —20°C. The
precipitate was dried and re-suspended in 20 µl of water.
Reverse transcription-polymerase chain
reaction and analysis of product
The RT-PCR assay for the detection of CSFV has been
previously described (5). In brief, the PCR primers are
derived from the p l 2 5 gene of the alfort and Brescia strains
(9,
11), i.e.
CSFV-1
nucleotides
5067-5087
and
5 ' -GCTCCTGGTTGGTAACCTCGG- 3", and CSFV-2 nucleotides 5 5 5 4 - 5 5 7 4 , 5 ' -TGATGCTGTCACACAGGTGAA- 3'.
The PCR protocol was conducted as follows: denaturation at
94°C for one minute, followed by 59°C for one minute for
primer annealing, and 72°C for two minutes for elongation. A
total amount of 10 µl from each reaction mix was analysed in
2 % agarose gel, stained with ethidium bromide and visualised
by transillumination.
Analysis by restriction endonucleases
Aliquots of 10 µl of the amplified RT-PCR mixtures were
digested with the restriction enzyme Ava II. The resulting
fragments were electrophoresed and visualised as described
above.
Spleens were harvested, as follows:
Virus isolation and immunoperoxidase assay
- 4 5 from sows
- 13 from market hogs
- 2 from three-week-old pigs.
Suspensions of porcine kidney (PK) 15 cells were inoculated
with 50 µl of 1 0 % tissue homogenate and seeded in duplicate
into 96-well plates. The cells were then incubated at 37°C for
4 8 hours in a 5 % C 0 incubator. The cells were subsequently
fixed with 2 0 % acetone for ten minutes and CSFV antigens
were detected using a monoclonal antibody, W H 3 0 3 (4).
CSFV-seronegative mouse ascites fluid was applied to the
2
Submandibular and mesenteric lymph nodes were collected
from adult pigs. To generate coded tissue panels, samples
were collected from eight- to ten-week-old piglets infected
676
Rev/, sci. tech. Off. int. Epiz., 17 (3)
duplicate set of wells to act as a control. The cells were then
washed. Bound anti-CSFV antibodies were detected with
horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G, and subsequent use of enzyme substrate,
hydrogen peroxide and chromogen,
3-amino-9-ethylcarbazole (1).
Table II
A n a l y t i c a l sensitivity of reverse transcription-polymerase chain
reaction and immunoperoxidase assays for the detection of c l a s s i c a l
s w i n e fever virus in infected tissues
Dilution
Results
Specificity of classical swine fever virus
reverse transcription-polymerase chain reaction
A total of 127 abattoir samples from different tissues namely: tonsils, spleens, submandibular lymph nodes,
mesenteric lymph nodes and kidneys - were tested by virus
isolation and immunoperoxidase assay (VI-IPA) and by
RT-PCR. All samples tested gave negative results by both
assays. Subsequently, 2 0 of those samples were chosen at
random and passaged in PK15 cells before being retested to
ensure that they were true negatives. The harvested cell
culture fluids were all found to be negative by both assays.
Analytical sensitivity of the assay
Virus titre (IU/ml)
RT-PCR
IPA
Undiluted
1.25x10"
+
+
1(T
1.25 x 1 0
+
+
+
+
+
-
1
irr
2
IO"
3
KT
4
IO"
5
IU
3
1.25X10
1.25x10'
2
1.25
1.25 x 1 0 -
1
-
-
-
: infectious units
RT-PCR : reverse transcription-polymerase chain reaction
IPA
: immunoperoxidase assay
A 10% (weight/volume) emulsion was produced from a classical swine fever virus-infected
tonsil. A ten-fold dilution series was then conducted. Total nucleic acid was isolated from each
dilution and RT-PCR was conducted as described in 'Materials and methods', above. A total
volume of 200 µl of virus suspension were used for the isolation of nucleic acid. The isolated
nucleic acid was re-suspended in 30 µl of water. Subsequently, 7 µl of the suspension were
used for RT-PCR. For IPA, 50 µl of virus suspension were used to infect porcine kidney 15 cells
obtained from infected tissue culture fluids diluted up to
10 -fold. At the same dilution, VI-IPA gave negative results.
The samples which gave positive results by VI-IPA were
diluted up to 10 -fold, ten-fold less than in the case of
RT-PCR. Taking into consideration the dilution factor,
RT-PCR could detect amounts as small as 0.1 infectious
particle per ml of fluid, whereas 1 infectious unit per ml could
be detected by VI-IPA. Similar experiments were performed
on 1 0 % tonsil tissue emulsions and the results are shown in
Table II. In infected tonsil tissues, amounts as small as
0.6 infectious unit per 100 mg of tissue could be detected by
RT-PCR, which represented a thousand-fold dilution,
whereas, in VI-IPA, sensitivity dropped ten-fold to 6
infectious units per 100 mg of tissue.
6
The analytical sensitivity of the assay in relation to VI-IPA was
determined by contrasting the detection of CSFV nucleic acid
by RT-PCR with that of CSFV antigen by VI-IPA. These tests
were performed on tonsil tissues collected from a pig infected
with the Standard strain, and on CSFV grown in cell culture
(Tables I and II). Serial dilutions of infected tissue culture
fluids were made in DMEM and subsequently tested for viable
virus by VI-IPA. The RT-PCR assay was conducted on nucleic
acid isolated from these dilutions. The results are shown in
Table 1. In regard to RT-PCR assays, positive results were
Table I
A n a l y t i c a l sensitivity of reverse transcription-polymerase chain
5
reaction and immunoperoxidase assays for the detection of classical
Diagnostic sensitivity of the assay
s w i n e fever virus in tissue culture fluids
The internal coded panels (shown in Tables III and IV) were
tested by both VI-IPA and RT-PCR. The first blind panel
Dilution
Virus titre (IU/ml)
RT-PCR
IPA
tested (Table III) consisted of eleven positive samples and one
Undiluted
2x1Q
NT
+
negative sample. The samples were from five animals, each of
10"
1
+
2
2x10
2x10"
+
IO"
+
+
KT
3
2x10
3
+
+
4
6
5
which was experimentally infected with one of three different
strains of CSFV, namely: the UK, Standard
and BAI
Nervous
isolates. All infected samples returned positive results on the
first round of RT-PCR. Nine of eleven samples gave positive
2x10
2
+
+
itr
•I - 6
2x10
1
+
results by VI-IPA without any amplification in PK15 cells. The
2
+
+
_
10"
2x10"'
-
-
and a kidney, returned positive results after passage in PK15
IO"
5
0
7
IU
RT-PCR
IPA
NT
:
:
:
:
infectious units
reverse transcription-polymerase chain reaction
immunoperoxidase assay
not tested
A ten-fold dilution series was conducted on both samples. Total nucleic acid was isolated from
each dilution and RT-PCR was conducted as described in 'Materials and methods', above. A
total volume of 200 µl of virus suspension were used for the isolation of nucleic acid. The
isolated nucleic acid was re-suspended in 30 µl of water. Subsequently, 7 µl of the suspension
were used for RT-PCR. For IPA, 50 µl of virus suspension were used to infect porcine kidney
15 cells
remaining two samples, tissues from a mesenteric lymph node
cells. All the samples in the second panel, twelve positive and
one negative, were correctly identified by RT-PCR in the first
round of testing, and all but one by VI-IPA upon initial
testing. The remaining sample, an infected ileum, tested
positive after a passage in PK15 cells. For the external panel,
both assays correcdy identified 18 out of the 2 0 samples,
giving complete agreement between the tests (Table V).
However, both RT-PCR and VI-IPA missed two samples,
tissues from the intestine and ileum, that originally gave
677
Rev. sci. tech. Off. int. Epiz., 17 (3)
Table III
positive
Comparative evaluation of reverse transcription-polymerase chain
samples, which returned positive results by RT-PCR, tested
reaction a n d i m m u n o p e r o x i d a s e assays for the detection of c l a s s i c a l
positive by VI-IPA only after passage in PK15 cells. To
s w i n e fever virus
The first internal blind panel w a s tested by RT-PCR and IPA, as described in
'Materials and methods', above
results.
Furthermore,
liver
tissue
confirm the RT-PCR results, the generated fragments were
fragments generated (data not shown) were as expected ( 6 ) ,
confirming the identity of the products.
Detection method
Sample
Strain
RT-PCR
IPA
1
Tonsil
UK isolate
+
+
1
SMLN
UK isolate
+
+
2
Spleen
ß4/
+
+
2
Kidney
BAI
+
3
MLN
Standard
+
3
Blood
Standard
+
4
MLN
Standard
+
4
Kidney
Standard
+
4
Tonsil
Standard
+
+
5
MLN
Standard
+
+
Tonsil
5
SMLN
Standard
+
+
Tonsil
6
Blood
Uninfected
-
-
Spleen
:
:
:
:
:
infected
isolated and digested with Ava II. The size and number of
Pig No.
RT-PCR
IPA
UK
SMLN
MLN
two
l a )
iB)
+
(b|
+
Table V
Comparative evaluation of reverse transcription-polymerase c h a i n
reaction and i m m u n o p e r o x i d a s e assays for the detection of c l a s s i c a l
s w i n e fever virus
A n external blind panel w a s tested by RT-PCR and IPA, as described in
'Materials and methods', above
+
+
(b)
+
Pig No.
Sample
Detection method
RT-PCR
IPA
Spleen
reverse transcription-polymerase chain reaction
immunoperoxidase assay
United Kingdom
submandibular lymph node
mesenteric lymph node
Lung
Lung
Liver
Liver
a| Samples were processed to mimic field conditions
Tonsil
b) IPA returned positive test results after one round of amplification in porcine kidney 15 cells
Spleen
Lung
Lung
Ileum
Table IV
Brain
Comparative evaluation of reverse transcription-polymerase chain
Kidney
reaction and i m m u n o p e r o x i d a s e assays for the detection of c l a s s i c a l
Intestine
s w i n e fever virus
Spleen
The second internal blind panel w a s tested by RT-PCR and IPA, as described
Tonsil
in 'Materials and methods', above
Spleen
Pig No.
Sample
Strain
1
Spleen
1
Lung
Detection method
RT-PCR
IPA
Wkisolate
+
+
Kidney
(//(isolate
+
3
Ileum
Standard
+
3
Tonsil
Standard
+
+
3
Ileum
Standard
+
+
4
Blood
Standard
+
+
4
Blood
Standard
+
+
the kappa statistic. The results showed that the two tests were
4
Spleen
Standard
+
+
in total accord.
4
MLN
Standard
+
+
5
Ileum
Standard
+
+
7
Spleen
New South
Wales
m
+
+
7
Tonsil
New South
Waies^
+
+
6
Blood
Uninfected
-
-
RT-PCR
IPA
UK
MLN
:
:
:
:
a) IPA returned positive test results after an amplification round in porcine kidney 15 cells
+
lal
+
RT-PCR : reverse transcription-polymerase chain reaction
IPA
: immunoperoxidase assay
reverse transcription-polymerase chain reaction
immunoperoxidase assay
United Kingdom
mesenteric lymph node
a) IPA returned positive test results after one round of amplification in porcine kidney 15 cells
b] Tissues were processed to mimic field conditions
b) Samples were originally positive
To assess the degree of agreement between the two tests, data
obtained from testing the three coded panels were analysed by
Discussion
Previous work on the RT-PCR of CSFV developed in the
laboratory of the authors (5, 6) indicated that the test is highly
specific. Testing was conducted for viruses other than CSFV
and all samples gave negative results. A limited number of
negative tissues were also tested, producing similar results ( 6 ) .
The work described in this report is a comparative validation
of RT-PCR and VI-IP assays using a larger number of tissues,
Rev. sci. tech. Off. int. Epiz., 17 (3)
678
both positive and negative. The results clearly reinforce
previous conclusions and support the use of the assay as a
detect CSFV was not due to an CSFV strain or tissue type. One
explanation could be that there were too few infectious
particles for detection by VI-IPA, and an amplification cycle
routine diagnostic test.
brings the amount up to detectable levels. Another possibility
The importance of the RT-PCR assay is underlined in the
comparison of its analytical sensitivity with that of VI-IPA. In
infected tissue culture fluids, VI-IPA is able to detect one
infectious unit; in infected tonsil tissue, sensitivity decreases
to approximately six infectious units. This could be due to
deterioration of the sample following storage for several
months or the effect of freezing and thawing on virus
infectivity. A ten-fold increase in sensitivity is observed when
RT-PCR is performed. The high analytical sensitivity achieved
by RT-PCR (less than 1 infectious unit per ml) has also been
noted in the case of other viruses ( 2 , 7, 1 5 ) .
is that the virus has to adapt to the cell line, provided by a
In terms of diagnostic sensitivity, the VI-IPA and RT-PCR tests
were in total agreement. All the samples were detected by
both assays when the internal panels were used, and both
assays missed two positive samples when the external panel
was tested. Two passages of the two false negatives did not
improve the results in either test. That would suggest that the
virus, present at one point in those samples, had been
destroyed. The samples - infected intestine and ileum tissues
- may have been improperly stored or handled, leading to
degradation of the infectious particles and the genome. The
results of the three coded panels together gave an overall
sensitivity of RT-PCR to CSFV of 9 5 % , the same sensitivity as
VI-IPA. However, in the latter, five samples required
amplification in PK15 cells before giving positive test results.
VI-IPA results will be known only after four days. Taking into
passage in PK15 cells, before antigen production reaches
detectable levels. Although a few positive tissues were missed,
all infected animals were correctly identified as most of their
tissues gave positive test results.
It is well documented that RT-PCR assays require less time
than most other tests (18, 2 1 ) , an important feature in
diagnostic virology. Similarly, in the work described in this
paper, a minimum of 4 8 hours is required for a VI-IPA to be
completed, whereas the result for an RT-PCR is known within
2 4 hours. Moreover, if an amplification round is required,
consideration the sensitivity of the assay and the time required
for completion of the test, RT-PCR would be a significant
addition to the tools available for the diagnosis of CSFV.
Acknowledgements
The authors are grateful to Y. Robinson of the Health of
Animals Laboratory, Canadian Food Inspection Agency,
St Hyacinthe, Quebec, for kindly providing the tissue samples
from abattoirs, and J . E . Pearson of the Diagnostic Virology
Laboratory, National Veterinary Services Laboratories, United
States Department of Agriculture, Ames, Iowa, for providing
CSFV-infected tissues.
The five samples were from four different tissues, harvested
from three different pigs which had been infected with three
strains of the virus. This would suggest that the failure to
•
Comparaison entre la technique de transcription
inverse-amplification en chaîne par polymérase et l'isolement
du virus pour la recherche du virus de la peste porcine classique
S. Vydelingum, T. Tao, K. Balazsi & R. Hecker
Résumé
Les auteurs évaluent la capacité d'une épreuve de transcription inverseamplification en chaîne par polymérase (RT-PCR) à déceler le virus de la peste
porcine classique, par comparaison avec l'isolement et la détection du virus selon
la méthode d'immunoperoxydase indirecte (VI-IPA). Pour déterminer la spécificité
de l'épreuve, des prélèvements ont été effectués sur 60 rates, 45 amygdales,
10 ganglions lymphatiques sous-mandibulaires, 8 ganglions lymphatiques
mésentériques et 4 reins, provenant de porcins d'âges divers abattus au Canada
(où la population porcine est indemne du virus de la peste porcine classique).
Tous les prélèvements ont donné des résultats négatifs par les deux techniques.
679
Rev. sci. tech. Off. int. Epiz., 17 (3)
VI-IPA et RT-PCR. En tout, 20 échantillons ont été passés dans des cultures
cellulaires de rein de porc 15 (PK15) et vérifiés à nouveau par les deux méthodes.
Ils ont tous donné des résultats négatifs, confirmant la spécificité (100%) des
épreuves.
Pour déterminer la sensibilité analytique des épreuves, une autre étude
comparée a été réalisée, utilisant le virus de la peste porcine classique obtenu en
culture cellulaire et à partir de tissus d'amygdales provenant d'un porc infecté.
Pour les tissus infectés comme pour les milieux de culture cellulaire liquides, la
sensibilité de la méthode RT-PCR était dix fois supérieure à celle de la technique
Vl-IPA. La méthode RT-PCR a ainsi permis de déceler 0,6 unité infectieuse pour
100 mg de tissus, un titre particulièrement faible, contre 6 unités infectieuses
détectés par la technique VI-IPA. De même, la méthode RT-PCR décelait jusqu'à
0,1 unité infectieuse par ml dans les milieux de culture liquides, contre 1 unité
infectieuse par ml détectée par l'épreuve VI-IPA.
Pour déterminer la sensibilité diagnostique, trois séries codées (deux internes et
une externe) de 45 échantillons provenant de 14 porcs ont été soumises à des
tests. La sensibilité diagnostique des deux techniques, VI-IPA et RT-PCR, a été de
100 % pour les deux séries internes. Les résultats de la série externe coïncidaient
également, à l'exception de deux prélèvements qui ont échappé à la RT-PCR
comme à la VI-IPA. Ces deux prélèvements donnaient toujours des résultats
négatifs après amplification en cellules PK15. Tous les résultats de la méthode
RT-PCR ont été obtenus lors d'une épreuve unique, alors qu'avec la technique
Vl-IPA, des résultats positifs n'ont été obtenus pour cinq prélèvements qu'après
amplification dans des cellules PK15. L'application de l'épreuve RT-PCR au
diagnostic de la peste porcine classique permettrait d'améliorer la détection du
virus et de réduire les délais d'obtention des résultats.
Mots-clés
Détection - Épreuve de l'immunoperoxydase indirecte - Transcription inverseamplification en chaîne par polymérase - Virus de la peste porcine classique.
Comparación entre una prueba de transcripción inversa-reacción
en cadena de la polimerasa y una de aislamiento vírico aplicadas a
la detección del virus de la peste porcina clásica
S. Vydelingum, T. Tao, K. Balazsi & R. Hecker
Resumen
Los autores evaluaron la capacidad de una prueba de transcripción
inversa-reacción en cadena de la polimerasa (reverse
transcription-polymerase
chain reaction, RT-PCR) para detectar el virus de la peste porcina clásica,
comparándola con el aislamiento y detección del virus por el método de
inmunoperoxidasa indirecta (VI-IPA). Para determinar la especificidad del ensayo
se analizaron muestras de 60 bazos, 45 amígdalas, 10 ganglios linfáticos
submandibulares, 8 ganglios linfáticos mesentéricos y 4 riñones procedentes de
cerdos de edades diversas sacrificados en mataderos canadienses (una
población libre del virus de la peste porcina clásica). Todas esas muestras
arrojaron respuesta negativa tanto a la prueba de aislamiento con detección por
VI-IPA como a la de RT-PCR. Tras pasar un total de 20 muestras por un cultivo de
células de riñon de cerdo 15 (PK15), se sometieron nuevamente esas muestras a
ambos ensayos, con resultados nuevamente negativos. De ahí se deduce una
especificidad del 100%.
680
Rev. sci. tech. Off. int. Epiz., 17 (3)
Para determinar la sensibilidad analítica del ensayo se llevó a cabo un estudio
comparativo similar, utilizando virus de la peste porcina clásica reproducidos en
cultivo celular y tejido amigdalar de un cerdo infectado por el virus. Tanto en el
caso del tejido infectado como en el del líquido del cultivo celular, la prueba de
RT-PCR resultó diez veces más sensible que la del aislamiento con detección por
VI-IPA. Mientras que la primera era capaz de detectar dosis ínfimas, de hasta
0,6 unidad infecciosa por 100 mg de tejido, el método VI-IPA alcanzaba sólo a
discriminar 6 unidades infecciosas. Análogamente, la prueba de RT-PCR podía
detectar 0,1 unidad infecciosa por ml de líquido de cultivo celular contra 1 unidad
infecciosa por ml en el caso del método VI-IPA.
Para determinar la sensibilidad de diagnóstico se analizaron tres paneles
codificados (dos internos y uno externo), que comprendían 45 muestras
procedentes de 14 cerdos. La sensibilidad de diagnóstico de ambas pruebas,
VI-IPA y RT-PCR, resultó ser de un 100% para los dos paneles internos. Los
resultados del panel externo también coincidían, con la salvedad de dos muestras
que ambos ensayos pasaron por alto. Tras amplificarlas en células PK15, esas dos
muestras siguieron dando resultado negativo. Mientras que todos los resultados
del método RT-PCR se obtuvieron aplicando una prueba única, en el caso del
método VI-IPA hizo falta una ronda de amplificación en células PK15 para obtener
resultados positivos para cinco de las muestras. La aplicación del ensayo de
RT-PCR al diagnóstico del virus de la peste porcina clásica haría posible una
mejor detección en un lapso menor de tiempo.
Palabras clave
Detección - Ensayo de inmunoperoxidasa indirecta - Transcripción inversa-reacción en
cadena de la polimerasa- Virus de la peste porcina clásica.
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