Download Nucleic acid hybridisation and polymerase chain reaction

Rev. sci. tech. Off. int. Epiz., 1993,12 (2), 405-423
Nucleic acid hybridisation and polymerase
chain reaction in the diagnosis of infectious
animal diseases
M. R O D R Í G U E Z and A . A . S C H U D E L *
Summary: The authors describe and summarise the use of nucleic acid
hybridisation and polymerase chain reaction (PCR) technologies in the
diagnosis of animal diseases.
PCR is a powerful biological tool which enables exponential
enzymatic
amplification in vitro of a given deoxyribonucleic
acid sequence. This
technique is currently used to study the molecular pathogenesis of many
infectious diseases and also for diagnosis. PCR is usually more sensitive than
conventional methods and does not require complex facilities, and will
therefore soon become the preferred technology of laboratory diagnosticians,
field veterinarians and health officers.
K E Y W O R D S : Animal health - Diagnosis - Polymerase chain reaction Probes.
INTRODUCTION
In the past, the diagnosis of infectious animal diseases has been h a m p e r e d by the
complexity of the technology, the facilities required and the amount of time needed for
detection and characterisation of most pathogens. With the advent of probe technology,
new methods are available to field veterinarians, diagnosticians and animal health
officers. T h e s e new m e t h o d s offer specificity and sensitivity equal to those of
conventional pathogen detection procedures, while possessing the advantage that they
are often able to produce results in a single day, thus providing the opportunity of taking
more effective control measures.
The presence of infectious agents in fluid or tissue samples can be revealed by
microscopic visualisation, or by detection of biological activity or structural components
(antigenic proteins and nucleic acids). The nucleotide sequence of the nucleic acid of
each pathogen is different to that of other micro-organisms and susceptible hosts. This
uniqueness forms the basis of two recently-developed diagnostic tools: nucleic acid
hybridisation and polymerase chain reaction (PCR). The characteristics and application
of these techniques in the diagnosis of infectious diseases of domestic animals are
considered below. Excellent earlier analyses of the technology should be consulted in
order to obtain a broader view (15,20,24,27,52,56,57,61,67).
* Institute of Virology, Centro de Investigación en Ciencias Veterinarias, Instituto Nacional de
Tecnología Agropecuaria, CC 77, Moron 1708, Argentina.
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TECHNICAL CONSIDERATIONS
Nucleic acid detection relies on the ability of complementary single-stranded (ss)
chains to react with each other. If one of these chains (probe) is labelled in some way, it
becomes possible to unveil the presence of the o t h e r chain in a hybridisation test.
Alternatively, using two short p r o b e s (primers) and a d e q u a t e reaction conditions,
minute quantities of a given pathogen can b e detected through P C R (Fig. 1).
Dot blot
Radioactive labelling
In situ
Non-radioactive
labelling
Polymerase
chain reaction
Results can be read in
agarose gels or by
hybridisation
Hybridisation
Amplification
Biotin
Digoxigenin
Sulfonylation
FIG.l
The dominant nucleic acid detection techniques currently available for the detection
of infectious agents
Extraction of nucleic acids
T h e initial step in the detection of nucleic acid is extraction of the acid from the
sample. Usually, ground tissue or fluid samples are i n c u b a t e d with d e t e r g e n t s and
digested with proteinase to release the nucleic acids which are separated from proteins,
lipids and cell debris by a phenol extraction. The nucleic acid is then recovered by an
ethanol precipitation and b o u n d to a solid support such as a nylon or nitrocellulose
membrane for hybridisation; alternatively, the nucleic acid is used directly for PCR.
Meticulous care should be taken when dealing with ribonucleic acid (RNA) targets,
to avoid degradation by ribonucleases (RNases). All glassware, water and materials
which are to be in contact with the sample should be treated. Autoclaved disposable
plasticware should be used, and disposable gloves should be worn at all times. Samples
should b e processed as quickly as possible and at low temperature. RNase inhibitors
such as vanadyl ribocomplexes and human placental ribonuclease inhibitor (RNAsin)
can help to avoid degradation (for greater detail, see 64).
In some instances, traditional extraction procedures have been successfully replaced
by more straightforward protocols. Examples of such protocols are the boiling of serum
for hepatitis B virus detection (F.E. Baralle, p e r s o n a l communication) and the
purification of cytomegalovirus ( C M V ) deoxyribonucleic acid ( D N A ) from urine
specimens by binding to glass powder (11).
Hybridisation
Classically, probes are produced by inserting sequences of the gene of interest into
the D N A of plasmid or bacteriophage vectors which can be propagated to obtain high
D N A yields (2). From the "library" of genomic sequences obtained, clones are selected
which suit the specific needs of a given test (specificity, sensitivity, ability to detect a
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broad spectrum of field strains). For diagnostic purposes, the purity of any nucleic acid
to be used as a probe is of prime importance, in order to avoid background signals which
would complicate the interpretation of results. Background noise due to non-specific
hybridisation to host or contaminant sequences is reduced by the excision of the cloned
fragment.
More recent a p p r o a c h e s for p r o b e p r o d u c t i o n are the use of synthetic
oligonucleotides, R N A probes and P C R products. The design of oligonucleotide probes
is based on available sequence information. Base-pair mismatches might occur when
such probes are used for the detection of field isolates, leading to reduced interaction
between probe and target. Several reports indicate that when highly-conserved regions
of the genome are selected and hybridisation conditions are optimised, oligonucleotide
probes can b e useful for viral detection (9, 10, 30). R N A p r o b e s , which are m o r e
difficult to handle because of the widespread presence of RNases, can be synthesised
in vitro in large amounts using specially-constructed recombinant plasmids such as those
containing the SP6 promoter. R N A probes are single-stranded (ss) and thus there is no
competing reassociation during hybridisation; they are therefore more sensitive than
double-stranded (ds) D N A probes (1). If the P C R has been optimised for a particular
pathogen, p r o b e s for detection of the p a t h o g e n can b e p r e p a r e d very easily using
labelled primers or deoxyribonucleotides linked to a biotin or digoxigenin residue.
Probes can be labelled with isotopes or non-radioactive markers such as biotin or
digoxigenin. Labelling involves replacement of a proportion of nucleotides in the probe
with labelled nucleotides (nick translation and random priming) or end labelling. The
nucleic acid can also be chemically modified (sulfonylated) in such a way t h a t it
becomes detectable by specific antibodies. Although P has been the label of choice
when sensitivity is of p r i m e i m p o r t a n c e , m a n y r e p o r t s (and the experience of the
present authors) indicate that P can be replaced, with important operative advantages,
by non-radioactive markers which do not constitute a health hazard to operators and
have a long shelf life.
3 2
32
Biotin has a high affinity for avidin and, when coupled to peroxidase or alkaline
phosphatase, serves to detect hybrids through a colorimetric reaction. Similarly, the
hapten digoxigenin is used to label probes which are detectable by means of specific
antibodies conjugated with an e n z y m e . F l u o r o c h r o m e s conjugated with biotin or
antibodies have also been used to label probes for in situ hybridisation, allowing the
location of target sequences in tissue sections and cell monolayers.
Hybridisation is carried out by incubating the labelled p r o b e , under a p p r o p r i a t e
conditions, with the m e m b r a n e in which the nucleic acids extracted from the sample
have been spotted. After several washes to remove unbound probe, the m e m b r a n e is
exposed to X-ray film (if the p r o b e is labelled with an isotope) or processed for
colorimetric visualisation of results if non-radioactive radiation is used (27).
Polymerase chain reaction
This technique, first described by Saiki and colleagues (63), enables the amplification
of a specific D N A sequence a million-fold or more. If the target is an R N A sequence, a
copy D N A has to be synthesised by reverse transcription before the PCR is carried out.
The reaction requires a cyclic, three-step process:
a) denaturation of D N A
b) annealing of primers
c) primer extension.
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Denaturation by heating at 93-95°C separates the two D N A strands. In the annealing
process, primers attach to complementary regions on either the left (5') or the right (3')
side of the sequence of interest. During primer extension, new strands of D N A are
produced by the addition of nucleotides (starting at the 3 ' end of the annealed primer),
to the unpaired D N A strand. This process takes place by the catalytic effect of a
thermostable D N A polymerase. The number of D N A strands doubles upon completion
of each cycle, and after 30-45 cycles millions of copies of the target sequence (known as
amplicons) are present in the test tube (Fig. 2).
2 DNA strands
(original)
(a)
(b)
4 DNA strands
(first cycle)
(c)
8 DNA strands
(second cycle)
(d)
Millions of copies
(30-40 cycles)
FIG.
2
The polymerase chain reaction process
The deoxyribonucleic acid (DNA) strands of the pathogen nucleic acid (a) are separated (denatured)
at 94°C, and the primers hybridise (anneal) to the complementary sequences at 37-60°C (b).
In a third step (extension), a thermostable DNA polymerase produces complementary strands at 72°C (c).
If this three-step process is repeated 30-40 times, an exponential accumulation of DNA copies occurs (d)
and the original target sequence is amplified millions of times
Selection of primers
Primers are synthetic ss oligonucleotides, the sequence of which is complementary
either to the left side of one strand of the target sequence (upstream primer) or to the
right side of the other strand ( d o w n s t r e a m p r i m e r ) . In o r d e r to select p r i m e r s for
detection of a particular pathogen, sequence data relating to the genome of the primers
must be available. Usually, the most suitable genomic portions for PCR tests intended
for detection of field strains are those which are conserved among isolates, although
some primer/template mismatches do not necessarily prevent amplification.
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After a genomic p o r t i o n is chosen, a search is c o n d u c t e d for pairs of 18-24 m e r
oligonucleotides with several characteristics:
- 40-60% G+C content
- absence of polypurines or polypirimidines
- absence of secondary structure
- absence of complementarity, particularly at the 3 ' end, which may p r o m o t e the
formation of an artifact called "primer dimer".
Computer programmes, available upon request, can help to perform this task (62).
Since the efficiency of the PCR is lower when long genomic fragments are amplified, the
selected primers should be less than 600 bases pairs (bp) apart.
Optimisation of PCR
The basic c o m p o n e n t s of a P C R r e a c t i o n are the p r i m e r s , deoxynucleotides
(dNTPs), a buffer containing magnesium chloride, a thermostable D N A polymerase
and target D N A . T h e concentration of reaction c o m p o n e n t s and t i m e / t e m p e r a t u r e
parameters must be adjusted for efficient amplification of specific targets. A suitable
starting point would be 200 µM of each d N T P , 0.2 µM of each p r i m e r , a C l M g
concentration of 1.5 mM, 2.5 units of enzyme and 10,000 copies of semi-purified target
DNA. T h e annealing t e m p e r a t u r e is initially defined by the characteristics of the
primers, while denaturation and extension are usually performed at 94°C and 72°C,
respectively. Thirty-five cycles with 90 sec for each step usually allow the visualisation of
a reaction product in agarose gels. The optimal concentration of Cl Mg, in the range of
1-6 mM, should b e subsequently d e t e r m i n e d . If smears are d e t e c t e d in the gel, in
addition to the expected fragment size, it might be necessary to raise the annealing
temperature, optimise the primer concentration or use other primers. Similar tests
should then be conducted with fewer target molecules and using artificial samples. In
this process, positive and negative controls should be included in each experiment.
Finally, to determine the specificity and sensitivity of the test, a significant number of
field and control samples should be examined and the results correlated with those
obtained using conventional techniques.
2
2
Amplicon detection
The P C R product (amplicon) is usually visualised as a distinct band in agarose gels
stained with ethidium bromide. T h e size of the amplicon, compared with molecular
weight standards, c o r r e s p o n d s to the length of the target s e q u e n c e defined by the
primers. The identity of the amplified fragment is confirmed by hybridisation with an
oligonucleotide p r o b e c o m p l e m e n t a r y to an internal area of the amplicon and the
presence of restriction enzyme sites. T h e hybridisation is p e r f o r m e d using a P C R
product spotted on membranes (dot blot), or after transfer of the nucleic acids present
in the agarose gel to a solid s u p p o r t ( S o u t h e r n blot). Restriction enzymes cut the
amplicons into fragments of defined size which are visible in agarose gels.
These procedures are relatively complex and are excessively time-consuming for the
clinical laboratory. A n interesting alternative which might allow the processing of many
samples simultaneously, and even a u t o m a t i o n , is the specific c a p t u r e of amplified
material onto solid phases, followed by colorimetric assay. This approach uses 96-well
microtitre plates to which a "capture p r o b e " is covalently attached (55). Amplicons
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g e n e r a t e d using biotin-labelled primers are t r a p p e d , and the p r e s e n c e of hybrids
detected by a streptavidin-peroxidase conjugate and a chromogenic substrate (35).
Laboratory procedure
False-positive reactions can arise due to contamination of a sample being examined
by PCR products of previous tests. Since millions of copies of target D N A are produced
in each positive r e a c t i o n and only a few are n e e d e d to p r o d u c e a false positive,
precautions should be taken to avoid this "carryover". The incorporation of positive,
negative and "no D N A " controls in each e x p e r i m e n t is essential to detect this
phenomenon. Good general laboratory practice should be followed to avoid this, along
with observance of the following guidelines (37):
- different laboratories and equipment should be used for pre- and p o s t - P C R
manipulations
- water, buffers, tips and tubes should be autoclaved, and work surfaces exposed to
ultraviolet light
- aliquots of reagents should b e p r e p a r e d daily for use in a P C R product-free
environment
- the D N A sample should be the last element added to each tube.
Critical experiments should be repeated and, whenever possible, more than one pair
of primers should be used to amplify disparate genomic fragments.
DIAGNOSTIC APPLICATIONS
While hybridisation tests serve some specific purposes quite well, relatively low
sensitivity and technical difficulties have limited the application of these tests in clinical
laboratories. This issue has been extensively reviewed by Knowles and G o r h a m (36).
Nevertheless, the high sensitivity of P C R , which is usually m o r e sensitive than
conventional methods, makes this technique a true candidate to replace many of the
procedures currently in use for the rapid detection of pathogens. The fact that very few
copies of the nucleic acid of a micro-organism are n e e d e d for rapid detection has
produced a m a r k e d increase in the n u m b e r of l a b o r a t o r i e s implementing P C R for
diagnostic purposes.
In contrast with conventional techniques, which demand a different procedure for
each pathogen, the implementation of P C R requires similar procedures and reagents
for all pathogens. After optimisation, P C R can be p e r f o r m e d in low-complexity
facilities using relatively low-cost equipment.
Detection of foot and mouth disease virus by PCR
The following provides an illustration of a strategy to develop and assess a P C R assay
for the detection of foot and mouth disease virus (FMDV). The gene coding for the viral
polymerase (located near the 3 ' end of the viral genome) was chosen for P C R because it
is highly-conserved among serotypes and knowledge of the sequence is available (45).
Oligonucleotides with suitable characteristics, including 50-60% G + C content and the
absence of secondary structure, were selected using a computer programme (62). The
reagent concentrations and t i m e / t e m p e r a t u r e p a r a m e t e r s were optimised using as
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template a plasmid in which the sequence of interest was inserted. The conditions for
reverse transcription were defined using purified viral R N A . Nucleic acid extraction
was performed following the method described by Meyer and colleagues (47). Results
were r e a d on agarose gels stained with e t h i d i u m b r o m i d e and confirmed by
hybridisation of the P C R products spotted on nylon membranes with a 30 mer internal
oligonucleotide labelled with digoxigenin (M. Rodríguez and colleagues, unpublished
findings).
In order to determine the sensitivity and specificity of the assay, experiments were
carried out using dilutions of viral stocks of serotypes A, O and C, artificial samples, and
bovine tissues taken at necropsy from experimentally-infected cattle (Tables I and II).
In addition, further proof of the specificity of the test was obtained by examining fifty
tissue samples (tongue epithelia, lung, soft palate, oesophagus, kidney and lymph node)
from uninfected and experimentally-infected cattle (P. M u r p h y and colleagues,
personal communication).
TABLE I
Assessment of the sensitivity of the polymerase chain reaction for the detection of foot
and mouth disease virus using dilutions of viral stocks of serotypes A, O and C
Viral stock
Serotype
Titre
(log LD /ml)
Sensitivity in gels
(LD )
6.90
7.04
7.04
6.80
1.90
0.27
0.27
0.19
10
No.
No.
No.
No.
644
547
622
578
Oi
Oi
A
87
50
50
LD :50% lethal dose for newborn mice
Results were read directly on agarose gels stained with ethidium bromide and
confirmed by hybridisation
50
The experimental data indicates that the test is specific and that sensitivity is at least
of the same order of magnitude as conventional methods when results are read directly
on agarose gels. I n c r e a s e d sensitivity is o b s e r v e d after hybridisation of t h e P C R
product. Many samples may be processed simultaneously in less than 24 h, and once the
test is optimised complex laboratory facilities are not required.
OTHER APPLICATIONS OF POLYMERASE CHAIN REACTION
FOR DETECTION AND CHARACTERISATION OF INFECTIOUS
ANIMAL DISEASE AGENTS
Viruses
African swine fever virus
Four P C R primers and one oligonucleotide probe were designed and synthesised to
amplify a 740 b p fragment corresponding to the conserved region of the g e n o m e of
African swine fever virus (ASFV). A specific 640 bp P C R product was amplified using
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TABLE II
Evaluation of the sensitivity and specificity of the polymerase chain reaction
test for the detection of foot and mouth disease virus using beef samples made
artificially (a), or beef and tongue epithelia taken at necropsy
from experimentally-infected
cattle (b)
Titre
(LD /0.25 ml)
Result of
PCR
Oi
Oi
Oi
54.00
5.40
0.54
Positive
Positive
Positive
Negative
A
O
c
1.98
15.70
1.98
Positive
Positive
Positive
Negative
A
O
C
1.00
1.24
1.00
Positive
Positive
Positive
Negative
Serotype
Sample
50
a)
Beef
Beef
Beef
Beef
homogenate
homogenate
homogenate
homogenate
+ stock no. 567
+ stock no. 567
+ stock no. 567
+ PBS
b)
Infected beef
Infected beef"
Infected beef "
Uninfected beef
(1)
(
(2)
Infected epithelium
Infected epithelium
Infected epithelium
Uninfected epithelium
<2)
<2)
For testing, 0.25 ml aliquots of a 1/10 homogenate were used and results were read directly on agarose
gels and confirmed by hybridisation
LD :50% lethal dose for newborn mice
PCR: polymerase chain reaction
PBS: phosphate-buffered saline
(1) three days post-infection
(2) sixteen days post-infection
50
oligonucleotides 1 and 5 as primers and assayed with templates extracted from organs
and plasma of ASFV-infected pigs. P C R was d e m o n s t r a t e d to be quicker and more
sensitive than conventional methods (73).
Avian endogenous leukaemia
virus
By the use of D N A amplification m e t h o d s , envelope genes of avian endogenous
leukaemia virus structures have b e e n partially sequenced, showing that subgroupspecific sequences of the endogenous loci were largely homologous with those of Rousassociated virus type O (60).
Avian
Polyomavirus
A P C R assay was developed for detection of an avian Polyomavirus, budgerigar
fledgling disease virus ( B F D V ) , using a single set of p r i m e r s c o m p l e m e n t a r y to
sequences located in the putative coding region for the B F D V VP1 gene. The assay was
very specific for B F D V and highly sensitive, being able to detect as few as twenty copies
of the virus. Using these methods, it was suggested that persistent infection may occur in
adult'birds, and further evidence was provided of possible in ovo transmission (58).
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Bluetongue
virus
PCR was adopted for the identification of a globally-conserved, serogroup-specific
nucleic acid sequence (210 bp) of bluetongue virus (BTV) R N A in B T V serotypes from
the United States of America ( U S A ) . Preliminary results indicate detection of target
RNA sequences at a level of less than 2 fg, equivalent to 7,500 viral particles (16). When
segment 7 primers (from BTV-ISA) were used, it was possible to detect as few as six
molecules of segment 7 ds R N A per sample and also to detect purified ds R N A from
different isolates of other serotypes. Positive results were obtained on red blood cells
from infected cattle containing as much as 16 T C I D , b u t not those containing
1.6 T C I D (81). Primers which encode virus reactive protein VP3, constructed using
sequences based on R N A segment 3, w e r e used to detect s e r o g r o u p s of B T V on
platelet, buffy coat and packed red cells of BTV-infected sheep (43).
5 0
5 0
Bovine
Coronavirus
PCR was used to synthesise ds and ss probes for detection of bovine Coronavirus
(BCV) using r e c o m b i n a n t plasmids as t e m p l a t e molecules. After fragment-specific
amplification by P C R , approximately ten viral genomes were detected by agarose gel
analysis (78). Diagnosis of B C V in 134 clinical samples by hybridisation was m o r e
effective with ss than with either ds probes produced by PCR, or a combination of six
recombinant plasmid probes containing non-overlapping sequences (79).
Bovine
herpesvirus-4
PCR was used and compared with hybridisation and cell-culture isolation of samples
from rabbits experimentally infected with bovine herpesvirus-4 (BHV-4). Virus was
detected by P C R in several organs (including n e r v o u s tissues) which w e r e found
negative by other techniques (50).
Bovine immunodeficiency
virus
Infection of embryonic bovine lung cells by bovine immunodeficiency virus was
monitored both by syncytia formation and by reverse transcription followed by PCR,
showing the advantages of the latter (33).
Bovine leukaemia
virus
Proviral bovine l e u k a e m i a virus ( B L V ) D N A was d e t e c t e d in lymphocytic and
tumour D N A at all stages of infection in cattle, by the use of a proviral B L V D N A
probe and amplification of the D N A (51). P C R amplification (gag-p24, env-gp51,
polymerase reverse transcriptase) was used to examine seropositive and seronegative
cattle for the p r e s e n c e of B L V D N A in p e r i p h e r a l blood m o n o n u c l e a r cells. This
protocol e n a b l e d the detection of one viral g e n o m e p e r 100,000 cells (49). W h e n
primers for the polymerase and px regions of B L V were used, as few as ten copies of
BLV D N A were consistently amplified. This technique was successfully used in a blind
study on blood mononuclear cells of eighteen cows (72).
Bovine
papillomavirus
Using PCR, bovine papillomavirus (BPV) D N A was detected in 70% of commercial
calf serum batches and 100% correlation was established with virus isolation (69). BPV
DNA was detected by P C R using a set of primers located in the E5 open reading frame
fitting BPV-1 and BPV-2, after extraction from formalin-fixed frozen sections of twenty
equine sarcoids. These results support the general view that B P V plays an important
role in equine sarcoids (74).
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Bovine
rotavirus
A full-length complementary D N A ( c D N A ) copy of the gene encoding the major
neutralising antigen of bovine rotavirus was amplified, enabling the detection of this
virus in infected faeces with 100,000 times greater sensitivity than the conventional
electropherotype method and a 5,000-fold increase in sensitivity over hybridisation (89).
Bovine virus diarrhoea virus
By the amplification and sequencing of the p l 2 5 coding regions of a base pair
homologous to the bovine virus diarrhoea virus (BVDV) biotype, cytopathogenic and
non-cytopathogenic Pe515 strains w e r e detected (18). Using a set of 20 bp primers
located in the conserved 3' region of the B V D V genome, it was possible to amplify a
205 bp target sequence from B V D V D N A . T h e amplification assay was sensitive
enough to detect one molecule of cloned B V D V cDNA, showing that the sensitivity of
the test is higher than 1 T C I D (42). P C R for B V D V was also shown to be ten times
more sensitive than virus isolation, by Vaniddekinge and colleagues (77). On the basis
of published sequence data on the N A D L (National Animal Disease Laboratory, USA)
strain of BVDV, primers from nucleotide 6322 to 7475 were extracted from serum and
white blood cells of persistently-infected heifers and used for P C R (8). Six 20 bp
sequences from across the viral genome were selected by homology analysis of the
published genomic sequence of the N A D L and Osloss isolates of B V D V . The probe
originating nearest the 5' end of the viral RNA, ND001, detected 86% of the cytopathic
and non-cytopathic viral isolates analysed (38). By using primers from the gp48 region
of the cytopathic N A D L strain, it was possible to detect B V D V infection in serum
samples of persistently-infected animals (6). D e g e n e r a t e oligonucleotide primers
designed on the basis of the sequence of two strains of B V D V and one strain of hog
cholera virus were used in the P C R to detect viral R N A in cells infected in vitro or in
circulating lymphocytes (83).
5 0
Caprine
arthritis-encephalitis
Caprine arthritis-encephalitis virus and maedi-visna virus were detected by PCR (90).
Distemper virus
Morbillivirus from Phoca vitulina was identified using P C R with c D N A derived from
rinderpest morbillivirus (25).
Equine arteritis virus
Three different primer pairs were used for reverse transcriptase P C R genomic R N A
amplification of clinical samples of equine arteritis virus, detecting as few as 600 plaqueforming units (PFU) per ml in seminal plasma (14).
Equine
herpesvirus-1
P C R was performed on infected and uninfected cultured cells and on 63 specimens
from 29 aborted equine foetuses. Results were evaluated by electrophoresis and dotblot hybridisation using an oligonucleotide probe labelled with biotin. Results were
obtained in 24 h and the close correlation with virus isolation results was established (3).
Unpurified D N A derived from cultures of equine foetal kidney cells infected with
equine herpesvirus ( E H V ) - l or E H V - 4 was amplified by P C R using o n e pair of
oligonucleotide primers. Restriction endonuclease digestion of the amplified segments
with P V u I I , followed by electrophoresis, revealed restriction fragment length
polymorphisms which enabled the two virus types to be differentiated (53).
415
Equine infectious anaemia virus
PCR was used to detect the presence of equine infectious a n a e m i a virus (EIAV)
genomes in peripheral mononuclear cells of horses experimentally infected with the
CL 22 strain of E I A V (87). Proviral sequences in the peripheral blood mononuclear
cells of three horses with acute E I A V infection were monitored using PCR. Provirus
was detected in the initial viraemic episode in each horse and during each of t h r e e
relapsing viraemic cycles. Following each viraemic e p i s o d e , provirus levels in the
peripheral monocytes decreased to less than one copy per 5 x 10 cells (54).
6
Feline leukaemia
virus
PCR was used to study the pathogenesis of experimental co-infection of cats with
feline influenza virus and feline leukaemia virus (FeLV) (76). P C R has also been used
for identification of FeLV infection on feline olfactory neuroblastoma (68).
Foot and mouth disease virus
Rapid and sensitive detection of F M D V in b o v i n e and p o r c i n e tissues was
successfully achieved by enzymatic R N A amplification of the polymerase gene. This
method was able to detect a m o u n t s smaller t h a n 1 T C I D when c o m b i n e d with
hybridisation to a labelled probe. No cross-reaction with twelve other viruses (including
enterovirus) was demonstrated (47).
5 0
Hog cholera virus
Total R N A isolated from hog cholera virus-infected tissue was reverse transcribed
and the resulting c o m p l e m e n t a r y D N A was amplified with a sensitivity of 10,000
T C I D . Sensitivity increased 1,000-fold when the P C R product was reamplified with a
set of nested primers (40).
50
Infectious avian laryngotracheitis
virus
The infectious laryngotracheitis virus (IALV) homologue of the herpes simplex virus
glycoprotein B (gB) gene was identified by PCR amplification of genomic IALV D N A (59).
Infectious bronchitis virus
A P C R technique was developed which p e r m i t t e d the typing of avian infectious
bronchitis virus isolates which caused nephritis (39).
Influenza
virus
In order to determine the persistence of influenza virus in ducks after oral infection,
PCR analysis of the haemagglutinine gene was used, showing no detectable R N A in
spleen samples (82).
Maedi-visna
virus
By using a multiple primer set which generates D N A segments with overlapping
cohesive termini, maedi-visna virus can be amplified, retained, and detected in infected
cells with greater sensitivity (by more than two orders of magnitude) than using existing
methods (26). A maedi-visna-like virus (designated EV-1) isolated from sheep showing
symptoms of arthritis and pneumonia was analysed by P C R in order to demonstrate the
degree of homology with known maedi-visna virus strains (65).
416
Malignant catarrhal fever virus (Alcelaphine
herpesvirus-1)
Two pairs of thirty nucleotide p r i m e r s w e r e selected, corresponding to the
nucleotide sequence of the genomic D N A of alcelaphine herpesvirus-1 (AHV-1) (the
causative virus of bovine malignant catarrhal fever). The primers were synthesised and
successfully used for P C R on crude cell lysates infected with AHV-1 (31). A fragment of
A H V - 1 D N A was subcloned into p V C 1 8 and s e q u e n c e d . T h e subclone hybridised
strongly to A H V - 1 D N A , weakly to A H V - 2 D N A and not at all to D N A from bovine
herpesvirus ( B H V ) - l , BHV-2 and BHV-4. A two-stage (nested) P C R diagnostic test
was devised, using a portion of the subcloned A H V - 1 D N A sequence. Five A H V - 1 and
A H V - 2 isolates w e r e d e t e c t e d identically and specifically by P C R , while the same
procedure failed to detect BHV-1, BHV-2 and BHV-4 viruses. By this technique, levels
of A H V - 1 as low as 0.01 T C I D were detected (34).
5 0
Pseudorabies virus (Aujeszky
virus)
Genomic sequences of Aujeszky virus (pseudorabies virus: PRV) were amplified by
PCR from cells of infected cultures, nasal cells and organs from acutely-diseased pigs,
as well as from organs of latently-infected pigs (5). W h e n used directly on disrupted
cells with twenty-five cycles, results w e r e o b t a i n e d in 1 h which w e r e equal in
specificity and sensitivity to conventional methods (32). Various aspects of the latency
of P R V in swine were investigated using in vitro nucleic acid amplification methods
based on PCR. Primers flanking the 156 bp region of the P R V gpII gene were annealed
to purified P R V D N A , as well as D N A isolated from trigeminal ganglia of swine
latently infected with P R V . Following amplification, 100 fg of P R V D N A were
visualised on stained gels and 1 fg (equivalent to six viral genome copies) was detected
when amplification was c o m b i n e d with blot hybridisation (41). W h e n blot
hybridisation of PCR-amplified D N A (gX and gII genes) was used, latent virus was
detected in seven of eight samples (44). A P C R protocol which specifically amplifies
sequences coding for the glycoprotein gp50 (a 217 bp sequence between 433 and 651 of
the gp50 gene) was able to consistently detect P R V in tissues of latently-infected swine.
Results were o b t a i n e d in 24 h by sampling tonsil tissues to investigate latent P R V
infection (85). Latent viral D N A was detected by P C R in trigeminal ganglia of all of
ten pigs which were necropsied 81 or more days after being infected intranasally with a
thymidine kinase-negative vaccine strain of P R V (80).
Rabies virus
P C R was used to study the primary multiplication site of a recombinant vaccinia
virus (VV) expressing the rabies virus G glycoprotein V V T G g R A B , in comparison
with the p a r e n t e r a l V V C o p e n h a g e n strain, after oral a d m i n i s t r a t i o n to foxes.
V V T G g R A B was d e t e c t e d in the tonsils of foxes tested after 24 h and 48 h, in the
buccal mucosa of foxes tested after 24 h and 48 h and in the soft palate of one of two
foxes tested after 24 h and one of three foxes after 48 h. Foxes were inoculated with
virus isolated from fox tonsils 24 h after oral administration (with or without cellculture amplification) to p e r f o r m back passages. N o virus could b e subsequently
isolated in either case. T h e innocuity of V V T G g R A B was also d e m o n s t r a t e d when
foxes w e r e inoculated with passaged virus (75). P C R was also used to study the
pathogenesis of rabies virus in a mouse model by inoculation of the maseter muscle,
showing that virus replication occurred on the trigeminal ganglia at early stages, while
at later stages (96 h post-infection) positive sense R N A was present in large amounts in
the maseter muscle (71).
417
Bacteria
Brucella abortus
A specific and sensitive P C R test was developed which was capable of detecting
0.1 pg of Brucella abortus D N A (less than 100 brucella cells) by the use of primers which
allowed the amplification of a 635 bp fragment of a 43 k D a outer m e m b r a n e protein
gene from B. abortus strain 19 (21).
Escherichia coli
A PCR has been developed to detect Escherichia coli with sensitivity and specificity
equal to conventional methods in the following substances: water (4), cheese (46), pig
stools (29) and minced meat (84). Similarly sensitive and specific detection of E. coli has
been achieved using oligonucleotide p r i m e r s containing inosine for enzymatic
amplification of different alleles of the gene coding for heat-stable toxin type I of
enterotoxigenic E. coli (13). Amplification of the mal B operon of E. coli A I I E strains
yielded a specific D N A fragment. The minimum detection limit was ten bacteria and the
PCR systems w e r e validated by testing twenty-seven E. coli strains of k n o w n
enterotoxigenic properties (13).
Leptospira spp.
A PCR was developed to detect leptospira. Primers were used to amplify a 631 bp
section of the 5 ' region of 16S ribosomal D N A . (28). When applied to the detection of
leptospira in urine samples from cows, as few as 5-10 leptospira per ml of urine were
detected (23).
Listeria monocytogenes
Five oligonucleotides w e r e used as p r i m e r s in the P C R for the amplification of
specific sequences from D N A of Listeria monocytogenes. This technique was used for
detection of listeria in food p r o d u c t s (7). Amplification of the alpha a n d b e t a
haemolysin gene was used for detection of L. monocytogenes D N A in cooked sausages
and milk, with a detection limit of ten bacteria per 10 ml of milk in 48 h (22). Variable
results were observed when P C R was used on cheese samples (84). With the aid of
PCR amplification, it was d e t e r m i n e d that the absence of a gene coding for
phosphatidylinositol is associated with the pathogenesis of L. monocytogenes (12).
Mycobacterium paratuberculosis
A 278 bp D N A probe (PCR 278) fragment was obtained by P C R amplification of the
5'region of IS900, an insertion element contained in the genome of M. paratuberculosis.
When used in conjunction with PCR, this probe could detect as little as 10 fg (equivalent
to two genomes) of M. paratuberculosis starting material (48).
Mycobacterium tuberculosis
Two oligonucleotides were synthesised and used as primers for P C R amplification.
A 396 bp fragment was specifically amplified from the Mycobacterium
tuberculosis
genome. N o amplification was observed from any of ten different
Mycobacterium
strains, including those belonging to the M. tuberculosis complex. The PCR product was
detected by dot blot hybridisation, even when as little as 10 fg of purified M. tuberculosis
DNA was used. A good correlation with isolation methods was established (17). Using
a 123 bp segment of IS6110, which is r e p e a t e d in the M. tuberculosis c h r o m o s o m e ,
a sensitive and specific assay system was devised for detection within 48 h of
418
M. tuberculosis on sputum samples (19). P C R was also found to be the most sensitive
test for the diagnosis of tuberculous meningitis (70).
Salmonella spp.
Specific primers and a magnetic immuno method (magnetic particles coated with
monoclonal antibodies) were used to amplify a 163 b p genomic fragment of Salmonella
typhimurium. By this method, a P C R amplification of 25 Salmonella strains but not of
19 other Enterobacteraceae was achieved. A sensitivity of 100 P F U was attained (88).
Other pathogens
Mycoplasma spp.
Primers designed on t h e basis of t h e sequences of t h e 16S ribosomal D N A genes
of Mycoplasma pneumoniae and M. genitalis enabled t h e development of a specific
and sensitive in vitro amplification assay system, which d e t e c t e d as few as 100
M. pneumoniae cells and 1,000 M. genitalis cells (66).
Toxoplasma spp.
Tissue samples from a b o r t e d foetuses were examined for t h e p r e s e n c e of
Toxoplasma by P C R amplification of the p30 gene of Toxoplasma. All samples were
shown to be infected (86).
*
* *
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