Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV)..[r]
Trang 1Investigation Journal of Veterinary Diagnostic
http://vdi.sagepub.com/content/6/1/44 The online version of this article can be found at:
DOI: 10.1177/104063879400600109
1994 6: 44
J VET Diagn Invest
Beverly J Schmitt, Osvaldo J Lopez, Julia F Ridpath, Judith Galeota-Wheeler and Fernando A Osorio Evaluation of PCR for Diagnosis of Bovine Viral Diarrhea Virus in Tissue Homogenates
Published by:
http://www.sagepublications.com
On behalf of:
Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
can be found at:
Journal of Veterinary Diagnostic Investigation
Additional services and information for
http://vdi.sagepub.com/cgi/alerts
Email Alerts:
http://vdi.sagepub.com/subscriptions
Subscriptions:
http://www.sagepub.com/journalsReprints.nav
Reprints:
http://www.sagepub.com/journalsPermissions.nav
Permissions:
http://vdi.sagepub.com/content/6/1/44.refs.html
Citations:
What is This?
- Jan 1, 1994 Version of Record
>>
Trang 2Evaluation of PCR for diagnosis of bovine viral
diarrhea virus in tissue homogenates
Beverly J Schmitt, Osvaldo J Lopez, Julia F Ridpath, Judith Galeota-Wheeler, Fernando A Osorio
Abstract Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV) Conventional virus isolation procedures showed the specimens contained BVDV The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting
Bovine viral diarrhea virus (BVDV) is an important using the polymerase chain reaction (PCR) tech-pathogen of cattle causing a variety of diseases that nique.1,2,5,6,10,11,15,23,24,26
range in severity from mild inapparent infections to Application of PCR in a diagnostic setting requires fatal mucosal disease.20,21 BVDV is classified within adaptation for use on clinical samples Previous reports the family Flaviviridae and is a member of the genus on the use of PCR for detection of BVDV have not Pestivirus The viral genome consists of a single-strand- addressed the problems associated with clinical
spec-ed nonpolyadenylatspec-ed RNA about 12.5 kb in length.8 imens The purpose of this study was to evaluate PCR Isolates of BVDV are divided into 2 biotypes, cyto- detection of BVDV in field specimens using primers15,22
that amplify sequences near opposite ends of the BVDV genome.
pathic and noncytopathic, based on effects in cell
cul-ture.25 Infections of cattle with noncytopathic BVDV
during early gestation may cause abortion or birth of
calves immunologically tolerant to the infecting
vi-rus 12,17 These calves are persistent carriers of BVDV
and can be a source of infection to the herd.4
Super-infection of persistently infected cattle with a
cyto-pathic BVDV may induce mucosal disease.4,7
Detec-tion of persistently infected cattle is important for
control of BVD In addition, contamination of fetal
bovine serum and other bovine products used in cell
culture work with BVDV presents a problem in
pre-paring veterinary biologics.3,19 Traditionally, virus
iso-lation with confirmation by immunofluorescence
has been the method used to detect BVDV in clinical
samples and in contaminated cell cultures and
biolog-ical products These techniques are time
consum-ing and require the use of cell culture Several reports
have described rapid diagnostic tests for BVDV
From the Department of Veterinary Science, University of
Ne-braska, Lincoln, NE 68583 (Schmitt, Lopez, Galeota-Wheeler, Oso-
rio), and the Virology Cattle Research Unit, National Animal
Dis-ease Center, Agricultural Research Service, USDA, Ames, IA 50010
(Ridpath) Current address (Schmitt): Diagnostic Virology
Labora-tory, National Veterinary Services Laboratories, Veterinary Services,
Animal and Plant Health Inspection Service, USDA, Ames, IA
Received for publication March 26, 1993
Materials and Methods
Preparation of clinical samples Tissue samples from
cat-tle with clinical signs of BVD were submitted to the Uni-versity of Nebraska Veterinary Diagnostic Center virology laboratory All samples came from the state of Nebraska between 1988 and 1990 The samples were prepared as fol-lows Minimum essential medium (MEM) with 2% horse serum was added to tissue at a ratio of 3:l and then ho-mogenized in a stomacher for 2-4 min The homogenate was centrifuged at 2,000 rpm for 30 min at 4 C, after which the supernatant was poured into 15-ml snap cap tubes After initial detection of BVDV by virus isolation, homogenates were preserved by freezing at -70 C
Cell cultures Tissue homogenates were inoculated onto
primary bovine lung cells and bovine turbinate cellsa that were free of BVDV Cultures were maintained in MEM sup-plemented with 10% horse serum in Leighton tubes Cell monolayers were stained for BVDV antigen using fluores-cein-labeled antibodya and examined by fluorescent micros-copy Tissue homogenates that were positive for BVDV were used for further analysis by PCR
Titration of homogenates Before the PCR was used, the
titer of infectious BVDV was determined using microtitra-tion plates and bovine lung cells For noncytopathic BVDV samples, serial dilutions of virus were inoculated onto bovine lung cells and then infected with a cytopathic BVDV (Singer 44
Trang 3Use of PCR in diagnosis of BVDV infections 45
strain) End point was determined by observing inhibition
of cytopathic effect?
Primers for PCR The primers used in this study are listed
in Table 1 Primer set 115 amplifies a 205-base segment that
contains a BstIII site The primers were synthesizedb and
purified by either polyacrylamide gel electrophoresis (PAGE)
or high-performance liquid chromatography (HPLC) A
sec-ond set of primers was used on samples that were not
am-plified by primer set 1 with the exception of 3 samples that
lacked sufficient quantity for testing These primers are
lo-cated at the 5' end immediately upstream of the open reading
frame 22
Extraction of BVDV RNA from tissue homogenates. A
100-µl volume of tissue homogenate was added to 450 µ1 of
STE buffer (100 mM NaCl, 50 mM Tris, 5 mM
ethylene-diaminetetraacetic acid [EDTA]; pH 8) in a microcentrifuge
tube Proteinase K was added at a concentration of 20 µg/
ml, the solution was incubated at room temperature for 10
min, and 6 µl of 10% sodium dodecyl sulfate (SDS, 0.2% wt/
vol) was added After incubation at 56 C for 20 min, phenol/
chloroform extractions were done, and the nucleic acid was
precipitated with 60 µl of 3 M sodium acetate (pH 5.2) plus
1 ml cold (-20 C) 100% ethyl alcohol The precipitated RNA
was pelleted by centrifugation for 15 min at 12,000 x g at
4 C, washed with cold 70% ethanol, and dried in a vacuum
desiccator
Reverse transcription (R T) After resuspension of the RNA
pellet in 9 µl of sterile distilled water, 1 µl (40 µmol) of each
primer was added and the sample was incubated for 5 min
at 70 C The sample was chilled on ice for 1 min and 3 µl
of deoxynucleoside triphosphatesc (7.5 µM), 4 µl Schimke
solution (10 mM dithiothreitol, 10 mM MgCl,, 70 mM KCl,
80 mM Tris; pH 8), 1 µl of reverse transcriptased (19.6 u/ml),
and 1 µl of RNase inhibitore (28 u/µl) were added The
re-sulting RT reaction mixture was incubated at 42 C for 2 hr
Reaction mixtures were then chilled on ice for immediate
testing or stored at -20 C
PCR Viral strains NADL and Singer were used as
pos-itive controls for PCR Negative controls consisted of MEM
with 1) 10% horse serum and gentamicin and 2) tissue
ho-mogenate from a gnotobiotic calf A mixture of PCR reagents
without template was used as control for detection of
con-tamination with extraneous DNA
A master mix was made of the following reagents: distilled
water, 58.5 µl; 10x buffer, 9.5 µl (15 mM MgCl2, 400 mM
Tris-HCl, 500 mM KCl, 1% gelatin); dNTPS, 16 µl of
equi-molar mixture of dNTPs (5 mM); 3 µl (40 µmol) of each
primer A 5-µl aliquot of RT reaction mixture was added;
the resulting mixture was heated to 95 C for 5 min and chilled
on ice Then 2.5 units of Taq polymerasee in 5 µl of 1 x buffer
(10 x Promega buffer diluted 1: 10) were added for PCR
am-plification A layer of 60 µl of Nujol mineral oilf was added
The PCR program consisted of 1 min at 95 C (denaturation),
1 min at 55 C (primer annealing) and 2 min at 72 C (primer extension) for 40 cycles Primer set 2 was used at a concen-tration of 15 µmol per reaction during both RT and PCR All other parameters remained as previously described
Nucleic acid hybridization. PCR products using primer set 1 were analyzed by electrophoresis in 2% agaroseg in TBE buffer (0.045 M Tris borate, 0.001 M EDTA) Gels were blotted onto a nylon membrane, denatured, and cross-linked
by UV light The hybridization reaction used a 30-base probe with the following sequence: ACCTAAACCGAAGCAGGT-TACCAAGGAAGC The probe was end labeled with 32P using T4 polynucleotide kinase.h Unincorporated label was removed by chromatography using a Sephadex G-50 spin column Prehybridization was done in 5 x SSPE-1 x BP (2% bovine serum albumin, 2% polyvinylpyrrolidone)-1%
sodi-um dodecyl sulfate (SDS) for 30 min at 55 C The hybrid-ization was performed using the same buffer with addition
of at least 4 x 106 cpm probe/ml for 0.5 hr at 55 C The nylon filter was washed in 1% SSPE-1% SDS 3 times for 5 min at room temperature and 1 time for 1 min at 55 C The filter was dried, exposed to radiographic film, and developed Analysis of the amplified product from primer set 2 was performed using a probe made from a 365-base pair segment
of the BVDV-Singer genome’ and cloned into the pBluescript
II KS +/- vectorj The probe aligned with bases 182-545
of the BVDV NADL genome Phage T3 polymerase with
32
P-UTP was used to radiolabel the probe
Prehybridization was for at least 4 hr at 55 C in hybrid-ization buffer consisting of 6 x standard saline citrate (SSC),
2 x Denhardt’s reagent, 0.1% SDS, and 100 u/ml denatured salmon sperm Hybridization with the same buffer was done
at 55 C overnight The blots were washed twice for 5 min at room temperature in 1 x SSC and 0.1% SDS followed by 2 washes for 30 min in 0.2 x SSC and 0.1% SDS at 55 C Autoradiography was performed by exposing the blot to ra-diographic film
Results
Titration of BVDV infectivity in clinical samples.
Fifty-five of 60 tissue homogenates had sufficient quan-tity to be tested for virus concentration In 23 ho-mogenates, the concentration of BVDV (either cyto-pathic or noncytocyto-pathic) was 101.7-103.3CCID,,/ml The concentration of BVDV in 32 homogenates was
< 101.7 CCID50/ml
PCR results using primer sets 1 and 2 Using PCR
primer set 1, 46/60 homogenates (77%) were positive for BVDV (Table 2) Forty-six isolates were positive
by PCR using the UNL primers, for a detection level
Trang 4of 77% (46/60) Primer set 2 allowed detection of BVDV
in 10/11 homogenates that were negative using primer
set 1 These complementary primers boosted the
de-tection level to 93% (56/60).
Discussion
Practical application of PCR for diagnosis of BVDV
requires that it be useful for field samples such as tissue
homogenates Consideration should be given to the
sensitivity of the test and ability of the primers to
identify most, if not all, BVDV strains In this study,
PCR was evaluated for direct detection of BVDV in
tissue homogenates from field cases of BVD Using 2
sets of primers, 55/60 tissue homogenates positive for
BVDV by virus isolation were positive by PCR The
PCR procedure used here required a single
amplifi-cation cycle and used primers derived from either the
5' noncoding region or the 3' coding region of the viral
genome.
In an earlier study, a PCR technique was evaluated
on tissue samples without cell culture passage.2 These
authors tested their set of nested primers targeting the
genomic region of gp48 on 8 organ suspensions
posi-tive for BVDV by virus isolation They were able to
detect BVDV in the suspensions only after second stage
amplification by PCR.
In the present study, primer set 2 detected 3 samples
that had detectable virus titer but were negative with
primer set 1 Primer set 2 may amplify a more
con-served area of the BVDV genome The genomic
di-versity of BVDV is well documented and presents a
challenge to researchers looking for a nucleic acid-based
test that will detect all isolates 13,14,22
Further work in the development of a diagnostic
PCR procedure for detection of BVDV should focus
on the selection of a single universal set or mixture of
primers that can detect all strains of BVDV Also,
re-sitivity of PCR as a diagnostic test Routine use of PCR in a diagnostic setting will require the use of non-radioactively labeled oligonucleotide probes in con-firmatory hybridizations of amplified products.
Acknowledgements
We thank Dr E Berry (North Dakota State University, Fargo, ND) for sequence information and M Palmer and D Reichenbach for their excellent technical assistance The use
of a particular manufacturer’s product does not constitute
an endorsement on behalf of the USDA
1
2
3
4
5
6
7
8
9
verse transcriptases that act as DNA polymerases dur-
ing both reverse transcription and PCR can streamline
the procedure by combining the 2 reactions in 1 tube.18 10.
Preventing the RNA destroying activity of RNases by
addition of RNAase inhibitor in sample preparation 11.
could also be an important step in increasing the
sen-Sources and manufacturers
National Veterinary Services Laboratories, Ames, IA
Genosys, The Woodlands, TX
Pharmacia, Alameda, CA
Life Sciences, St Petersburg, FL
Promega, Madison, WI
Perkin-Elmer, Norwalk, CT
FMC, Rockland, ME
Biolabs, Beverly, MA
Dr E Berry, North Dakota State University, Fargo, ND Stratagene, La Jolla, CA
References
Alansari H, Brock KV, Potgieter, LND: 1993, Single and dou-ble polymerase chain reaction for detection of bovine viral di-arrhea virus in tissue culture and sera J Vet Diagn Invest 5:148-153
Belak S, Ballagi-Pordany A: 1991, Bovine viral diarrhea virus infection: rapid diagnosis by the polymerase chain reaction Arch Virol Suppl 3:181-190
Bolin SR, Matthews PJ, Ridpath JF: 1991, Methods for de-tection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus J Vet Diagn Invest 3: 199-203
Bolin SR, McClurkin AW, Cutlip RC, Coria MF: 1985, Severe clinical disease induced in cattle persistently infected with non-cytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus Am J Vet Res 46:573-576
Boye M, Kamstrup S, Dalsgaard K: 1991, Specific sequence amplification of bovine viral diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid Vet Mi-crobiol 29:1-13
Brock KV: 1991, Detection of persistent bovine viral diarrhea virus infections by DNA hybridization and polymerase chain reaction assay Arch Virol Suppl 3: 199-208
Brownlie J, Clarke MC, Howard CJ: 1984, Experimental pro-duction of fatal mucosal disease in cattle Vet Rec 114:535-536
Collett MS, Anderson DK, Retzel E: 1988, Comparisons of the pestivirus bovine viral diarrhoea virus with members of the Flaviviridae J Gen Virol 69:2637-2643
Gillespie JH, Madin SH, Darby NB: 1962, Cellular resistance
in tissue culture, induced by non-cytopathic strains, to a cyto-pathic strain of virus diarrhea virus of cattle Proc Soc Exp Biol Med 110:248-250
Hertig C, Pauli U, Zanoni R, Peterhans E: 1991, Detection of bovine viral diarrhea (BVD) virus using the polymerase chain reaction Vet Microbiol 26:65-76
Hooft van Iddekinge BJL, van Wamel JLB, van Gennip HGP, Moormann RJM: 1992, Application of the polymerase chain
Trang 5Use of PCR in diagnosis of BVDV infections 47
reaction to the detection of bovine viral diarrhea virus infections
in cattle Vet Microbiol 30:21-34
12 Kahrs RF: 1973, Effects of bovine viral diarrhea on the
de-veloping fetus J Am Vet Med Assoc 163:877-878
13 Kwang J, Littledike ET, Bolin SR, Collett MS: 1991, Efficiency
of various cloned DNA probes for detection of bovine viral
diarrhea viruses Vet Microbiol 28:279-288
14 Lewis TL, Ridpath JF, Bolin SR, Berry ES: 1991, Detection of
BVD viruses using synthetic oligonucleotides Arch Virol 117:
269-278
15 Lopez OJ, Osorio FA, Donis RO: 1991, Rapid detection of
bovine viral diarrhea virus by polymerase chain reaction Clin
Microbiol 29:578-582
16 Maniatis T, Fritsch EF, Sambrook J: 1982, Molecular cloning:
a laboratory manual Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY
17 McClurkin AW, Littledike ET, Cutlip RC, et al.: 1984,
Pro-duction of cattle immunotolerant to bovine viral diarrhea virus
Can J Comp Med 48:156-161
18 Myers TW, Gelfand DH: 1991, Reverse transcription and DNA
amplification by a Thermus thermophilus DNA polymerase.
Biochemistry 30:7661-7666
19 Nuttall PA, Luther PD, Stott EJ: 1977, Viral contamination of bovine feotal serum and cell cultures Nature 266:835-837
20 Olafson P, MacCallum AD, Fox FH: 1946, An apparently new transmissible disease of cattle Cornell Vet 36:205-213
21 Ramsey FK, Chivers WH: 1953, Mucosal disease of cattle North Am Vet 34:629-633
24 Schroeder BA, Balassu-Chan TC: 1990, Specific sequence am-plification of bovine viral diarrhea virus nucleic acid Arch Virol 111:239-246
22 Ridpath JF, Bolin SR, Katz JB: 1992, Comparison of nucleic acid hybridization and nucleic acid amplification based on con-served sequences from the 5' noncoding region for detection of bovine viral diarrhea virus J Clin Microbiol (in press)
23 Roehe PM, Woodward MJ: 1991, Polymerase chain reaction amplification of segments of pestivirus genomes Arch Virol Suppl 3:231-238
25 Underdahl NR, Grace OP, Hoerlein AB: 1957, Cultivation in tissue-culture of cytopathic agent from bovine mucosal disease Proc Soc Exp Biol Med 94:795-797
26 Ward P, Misra V: 1991, Detection of bovine viral diarrhea virus using degenerate oligonucleotide primers and the poly-merase chain reaction Am J Vet Res 52:1231-1236