Development of a One Step RT-qPCR Assay for the Identification of Ruminant Pestivirus

1. LINEAR RANGE AND ANALYTICAL SENSITIVITY EVALUATION OF CLINICAL CASES
Fig 2. Detection of BVDV/BDV was achieved in nearly almost the different evaluated type of samples (A), with most of
positives cases recorded in the reproductive swabs (23%) and the blood/serum samples (20%) (B). Other samples
included milk, semen and ear notches, positive cases were recorded only en the ear notches samples.
Fig 1. Reportable range for the EXOPOL oneMIX BVDV/BDV assay. Every replicate for standard dilutions over
102 copies/rxn resulted positive with Cq values ranging from 34.8 to 35.6 (A). Log. 10 fold serial dilutions of a
specific positive control were used to perform a standard curve; each dilution was evaluated five-fold (B).
ANALYTICAL SPECIFICITY
DEVELOPMENT OF A ONE-STEP RT-qPCR ASSAY
FOR IDENTIFICATION OF RUMINANT PESTIVIRUS
Benito A.A.; Arnal J.L.; Serrano J.D.; Baselga R.
Exopol S.L.; Zaragoza, Spain. abenito@exopol.com
Development of diagnostic assays should consider the broad genetic diversity of pestiviruses in order to obtain adequate tools for the control and erradication
programs of these diseases. In this study, our one step RT-qPCR assay detected properly a wide panel of ruminant pestiviruses strains obtained from the FLI; the
National Institute for Animal Health of Germany and responsible for the animal disease control programs in this country.
The oneMIX BVDV/BDV assay showed an excellent specificity and a good level of sensitivity with a low limit of quantification of 102 copies/reaction. These results
suggest that our assay could be used as an specific and sensitive test for the routine diagnosis of ruminants pestiviruses in different kind of samples including fetal
tissues, lungs, swabs, small intestine, feces and others.
Introduction
Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus
(BDV) are relevant pathogens for livestock industries
worldwide. These ruminant pestiviruses can cause significant
economic losses, mainly by reproductive diseases but also by
respiratory and enteric processes1,2. Additionally; BVDV and
BDV have been divided in several genotypes or even sub-
genotypes3,4, such diversity could complicate the proper
identification of these pathogens.
RT-qPCR is currently accepted as a reliable diagnostic tool for
several animal diseases; however, its complexity and high
relative prices have limited its use as a routine diagnostic test5.
In this study, we propose a novel RT-qPCR assay for a rapid,
and reliable diagnosis of pestivirus in domestic ruminants.
EXOPOL S.L.U.
Pol. Río Gállego, D-8. 50840
San Mateo de Gállego, Zaragoza. SPAIN.
Tel: +34 976 694 525 exopol@exopol.com
www.exopol.com
A single step RT-qPCR assay to detect BVDV (BVDV-1, BVDV-2) and BDV was designed targeting
the highly conserved 5’UTR region. A previously validated assay to detect β-actin gene6 was used as
an endogenous control (EC). All necessary reagents for a duplex RT-qPCR were included in a
single-tube reaction (oneMIX) and stored at -20°C until use. A synthetic and specific positive control
(PC) was designed and quantified to be used with this assay.
Setup: Only need to add 15µl of the
oneMIX and 5µl of the RNA/PC sample.
Run the qPCR: Amplification was performed in a StepOne Real Time PCR System (A.B.).
Probes were labeled for detection of the pathogen in FAM channel and EC in HEX/VIC channel
Reverse
transcription
RT enzyme
denaturation
cDNA amplification (45 cycles)
Species Strain name
subtype /
genotype
Cq values of quantificationb
FLI assayc
EXOPOL assay
BVDV-1 NADL 1a 27,3 29,1
BVDV-1 Paplitz 1b 27,6 28,1
BVDV-1 PI809 1d 27,9 27,9
BVDV-1 NC3807-1251/1 1e 28,2 28,5
BVDV-1 Egbert 1f 29,2 29,8
BVDV-1 BO806-17 1g 28,4 27,5
BVDV-1 BO807-3 1h 28,5 26,1
BVDV-1 NCP-2508-FCS 1c 29,2 31,8
BVDV-1 Böhni 1k 29,9 31,8
BVDV-1 NC3807-8757 1x 28,1 26,7
BVDV-2 8644 2a G 28,6 30,1
BVDV-2 Bure 2a US 28,3 32,3
BVDV-2 Walter 2b 28,4 30,7
BVDV-2 PO1600 2c 28,0 31,6
BDV Moredun 1 28,7 27,5
BDV Rudolph 2 28,0 28,6
BDV Gifhorn 3 28,5 32,5
BDV Isard 4 28,0 27,9
Pestivirus Hobi atypical 28,8 30,9
Pestivirus Giraffe H138 atypical 27,7 Neg
RT-qPCR Design
Validation of the EXOPOL oneMIX BVDV/BDV assay
a The EPIZONE Pestivirus-Reference RNA panel from FLI (Germany) was gently provided by Dr. Hoffmann.
b Threshold cycle (Cq) for approx. 2.0E+03 copies/µl.
c Data provided with the reference panel, the quantification assay is published in Hoffmann et al. 2006, Gaede et al. 2005.
Bacteria
Arcanobacterium pyogenes Leptospira interrogans (serovar Pomona)
Birbistenia trehalosi Listeria monocytogenes
Campylobacter fetus veneralis Listeria innocua (CECT 910)
Chlamydia pecorum (ATCC VR1575) Mannheimia haemolytica
Chlamydophila abortus Mycoplasma agalactiae
Clostridium perfringes Mycoplasma bovis
Clostridium difficile (CECT 531) Mycobacterium avium ss. paratuberculosis
Clostridium sporogenes (CECT 485) Pseudomona aeruginosa
Enterococcus feacalis (ATCC 29212) Salmonella enterica (CECT 4396)
Escherichia coli (ATCC 25922) Staphylococcus aureus (CECT 239)
Corynebacterium bovis Shigella flexneri (CECT 585)
Corynebacterium pseudotuberculosis Streptococcus agalactiae
Coxiella burnetti Streptococcus dysgalactiae
Dichelobacter nodosus Streptococcus uberis
Fusobacterium necrophorus Yersinia enterocolítica ( CECT 4055)
Virus Parasites
Bovine Herpesvirus 1 Cryptosporidium parvum
Bovine Respiratory Sincitial Virus Neospora caninum
Bovine Parainfluenza 3 virus Toxoplasma gondii
Rotavirus type A Theileria annulata
Coronavirus Bovino Tritrichomona foetus
A Pestivirus-reference RNA panel from the Friedrich Loeffler Instituta was used
fin this validation. A total of 19 ruminant pestivirus strains including: ten BVDV-1,
four BVDV-2, “Hobi-like” pestivirus and four BDV, resulted positive to this assay;
as detailed in table below.
A specificity panel including several related pathogens and biological
contaminants of the environment were also evaluated for analytical specificity.
Strains were obtained from EXOPOL strain bank, ATCC or CECT (Spanish Type
Culture Collection). None of these microbiological agents resulted positive.
Linear regression analysis demonstrates a dynamic range of quantification from
102 to 109 copies/rxn, with a R2 values of 0.99 and a slope of -3.479 that verifies
the linearity through the tested range (Fig.1). In the intra-assays tests the
coefficient of variation (CV) ranged from 0.72% to 9.27%, obtaining higher CV in
the lowest copy number dilution (102 copies/reaction).
A total of 272 clinical cases received in our lab for diagnosis of Pestivirus
infection (bovine=170, ovine/caprine=102) were evaluated. BVDV/BDV was
detected in 17.1% (29/170) of cattle and 16.7% (17/102) of sheep; but none in
goat samples. Tested samples included: fetal tissue/placenta, reproductive
swabs, feces/small intestine, lungs, blood/serum and other different samples.
REFERENCES
1. R. Krametter-Froetschera, M. Duenserb, B. Preylera, A. Theinera, V. Benetkac, K. Moestlc, W. Baumgartnera. 2010. Pestivirus infection in sheep and goats in West Austria. Veterinary Journal, Vol. 186 (3): 342–346.
2. S.R. Lanyon, F.I. Hill, M.P. Reichel, J. Brownlie. 2013. Bovine viral diarrhoea: Pathogenesis and diagnosis. Veterinary Journal, Vol 199 (2) 201–209.
3. K. Stahl, M. Beer, H. Schirrmeier, B. Hoffmann, S. Belák, S. Alenius. 2010. Atypical “HoBi-like” pestiviruses recent findings and implications thereof. Veterinary Microbiology, Vol. 142 (Issues 1–2): 90–93.
4. M. Giammariolia, S.A. La Rocca, F. Steinbachb, C. Casciaria, G. Mario De Mia. 2011. Genetic and antigenic typing of border disease virus (BDV) isolates from Italy reveals the existence of a novel BDV group. Veterinary Microbiology,
Vol. 147(Issues 3–4): 231–236.
5. E.J. Dubovi 2013. Laboratory diagnosis of bovine viral diarrhea virus. Biologicals 41 (2013) 8-13.
6. A.A. Benito, J.L. Arnal, E. de Tomas, J.D. Serrano, A. Villa. 2013. Internal controls for reliable results in real-time PCR diagnostic assays. WAVLD Berlin 2013 Abstract Book.
Conclusions
A B
Others
Blood/serum
Lungs
Feces/small
intestine
Reproductive
swabs
Fetal
tissues/Placenta
0 25 50 75 100
23
25
26
51
56
91
1
5
4
8
13
15
Numberof clinical cases
Positives
Total
A 4,3%
20,0%
15,4%
15,7%
23,2%
16,5%
Other samples Blood/serum
Lungs Feces/small intestine
Reproductiveswabs Fetal tissues/Placenta
B