A TaqManR RT-PCR Assay for the Detection of Feline calicivirus
Ashok K. Tiwari,
Kay S. Faaberg
Sagar M. Goyal
Feline calicivirus (FCV) is one of the most common
pathogens of cats causing upper respiratory tract infections. A real time
RT-PCR assay was developed for the rapid detection of FCV. A set of probe/primers
was designed to amplify a region of 151 bp based on the conserved region
of 7 FCV strains (FCV 255, 2280, NADC, F9, KCD, CF I and Urbana) by sequence
comparison. The assay was found to be more sensitive than virus isolation
and was linear over a wide range of template concentrations.
Feline calicivirus (FCV) is a single stranded RNA virus belonging
to the family Caliciviridae and is a common cause of upper respiratory
tract infection in cats (Gaskell and Dawson, 1994). In addition, FCV may
also cause conjunctivitis, stomatitis, enteritis and lameness (Baulch-Brown
et al., 1997). Infections caused by strains of low virulence are
usually restricted to oral mucosa, nasal passages and conjunctiva while
those by virulent and pneumonic strains show signs of severe respiratory
infection. Mortality is high in the presence of secondary bacterial infections
(Murphy et al., 1999). Recovered cats can serve as carriers of
the virus and are important in the maintenance and dissemination of infection
in domestic and wild cat populations. Harbour et al. (1991) reported
virus isolation rates of 20% from oropharyngeal swabs collected from domestic
cats without any breed or sex variation. A similar rate of high FCV carriage
was reported from a cat shelter and purebred cattery in California (Tenorio
et al., 1991). Although the duration of shedding is variable, approximately
50% of the infected cats cease virus shedding by 75 days post-infection
although individual cats may shed virus for up to 2 years (Barlough, 1992).
Clinical diagnosis of FCV is based on history and clinical signs but
definitive diagnosis requires virus isolation in cell cultures and/or
serology. Since virus isolation procedures are time consuming and expensive,
tests based on nucleic acid amplification (e.g., reverse transcription-polymerase
chain reaction or RT-PCR) have been developed. However, these tests have
been hampered by difficulty in designing oligonucleotide primers that
are able to amplify nucleic acids from all different strains of FCV (Sykes
et al., 1998). In addition, PCR products need to be detected by
gel electrophoresis or Southern blotting thus increasing the time and
expense in the specific detection process. Recently, Marsilio et al.
(2005) reported on a nested PCR (nPCR) for the detection of FCV. Although
more specific than RT-PCR, nPCR requires the use of two sets of primers
and two cycles of PCR followed by electrophoretic separation of bands,
which again increases the time as well as cost of diagnosis.
Recently, real time-PCR has become a method of choice for pathogen detection
because it allows real time quantification of PCR products, thus eliminating
the need for any post-amplification steps (Leutenegger, 2001). Fluorogenic
PCR assays have been successfully used in amplification and detection
of various bacterial and viral pathogens (Batt, 1997). Gut et al.
(1999) described a one-tube fluorogenic, RT-PCR for the quantification
of feline corona viruses. Schweiger et al. (2000) reported the
development of a TaqManR RT-PCR for typing and subtyping of
influenza viruses in the human clinical samples. This test was more sensitive
than viral isolation as it could detect up to 0.1 TCID50 of
the virus. The TaqMan assay employs 5 → 3exonuclease activity of
Thermus aquaticus (Taq) DNA polymerase to hydrolyze an internal
TaqMan probe labeled with a florescent reporter dye (FAM) and a quencher
dye (TAMRA). The most striking advantage of TaqMan assay over conventional
RT-PCR is the ability to save time as it allows real time quantification
of amplification products. In this report, we describe the development
of a TaqManR assay for the detection of FCV in clinical samples.
MATERIALS AND METHODS
Viruses and Cells
This study was done at the Veterinary Diagnostic Laboratory, University
of Minnesota, Saint Paul, MN. All seven FCV strains (FCV 255, 2280, NADC,
F9, KCD, CF I and Urbana) used in this study were obtained from Dr. John
Neill, National Animal Disease Center, Ames, Iowa. Monolayers of Crandell-Reese
feline kidney cells (CRFK) were used to propagate and titrate FCVs. Briefly,
the cells were grown in Eagles minimal essential medium (MEM; Celox,
St. Paul, MN) supplemented with 8% fetal bovine serum, penicillin (100
U mL-1), streptomycin (100 μg mL-1), fungizone
(1 μg mL-1), 15 mM HEPES and 5 mg mL-1 lactalbumin
hydrolysate. Confluent monolayers of CRFK cells were inoculated with the
virus and after adsorption for 1 h, the cells were incubated in the maintenance
medium (MEM without fetal bovine serum) at 37°C until the appearance
of cytopathic effects (CPE), usually within 2-3 days post-infection. The
viruses were harvested when CPE was observed in 60-80% cells. For virus
harvesting, the cultures were frozen and thawed twice followed by centrifugation
at 2500 x g for 10 min at 4°C. Clarified supernatant was aliquoted
in 1 mL amounts and stored at -70°C until further use. For virus titration,
serial 10 fold dilutions of viruses were prepared in maintenance medium
and inoculated in 96 well plates containing monolayers of CRFK cells.
Virus titers were calculated by the method of Reed and Muench (1938).
Viral RNA was prepared from 140 μL of cell-free extract using
the Viral RNA extraction kit (Qiagen, Valencia, CA) according to the manufacturers
instructions. RNA was eluted from silica columns in 60 μL of nuclease-free
water and stored at -70°C until further use.
Primer and Probe Design
Oligonucleotide primers were derived from conserved regions identified
by aligning the sequences of capsid protein gene of 7 FCV strains. Details
of strains used and the conserved regions are given in Table
1. Nucleotide sequences were retrieved from GenBank (http://www.ncbi.nlm.nih.gov)
and aligned using ClustalX method to determine the conserved region. Primers
and probe were designed using Primer Express software and were specific
to FCV 255 (Table 2). Specificity of both primers and
probe for other FCV strains was determined by BLAST analysis.
Real Time RT-PCR
Real time RT-PCR (rRT-PCR) was done using the TaqMan one step RT-PCR
Master Mix kit (Applied Biosystems, CA). Each reaction mixture consisted
of 12.5 μL of 2X Master Mix; 0.625 μL of 40X Multiscribe Mix;
0.4 μM of each primer; 0.8 μM of TaqMan probe; 5 μL of
template RNA; and 2.875 μL of water to make a final volume of 25
μL rRT-PCR was performed in 96 well plates (Applied Biosystems, CA)
using the ABI PRISM 7900 sequence detection system.
||Sequence alignment of different Feline calicivirus (FCV)
strains showing homologous region
||Nucleotide sequence of probe and primers used in real
Thermocycling conditions used were: reverse transcription (RT step)
at 48°C for 30 min; AmpliTaq Gold activation at 95°C for 10 min
and 50 cycles of PCR consisting of denaturation and annealing at 95°C
for 15 sec and 50°C for 1 min, respectively.
Specificity and Sensitivity of Assay
The sensitivity of the TaqMan assay was determined by testing RNA
extracts from serial 10 fold dilutions of FCV 255 (initial titer 1.45x109
TCID50 mL-1) as a template. For standard curve,
serial 10 fold dilutions of RNA extracted from undiluted virus were made
in DNase and RNase-free water and then used as a template in the TaqMan
assay. The detection threshold was based on the lowest concentration of
viral RNA which could be detected and remained within the linearity of
the standard curve. Specificity of the primers and probe was evaluated
using CHV (Canine herpes virus) and FPV (Feline panleukopenia
Samples of trachea, spleen, lung and regional lymph nodes are routinely
submitted to the Minnesota Veterinary Diagnostic Laboratory for the detection
of FCV. For isolation of FCV, tissue homogenates from these samples are
inoculated into CRFK cells and observed for development of CPE for up
to 7 days. Samples positive for CPE are examined by negative contrast
electron microscopy (Goyal et al., 1987). A total of 20 such samples
were tested with rRT-PCR and the results were compared with conventional
virus isolation method.
Analytical Specificity of rRT-PCR
The designed probe/primer set was able to amplify target viral RNAs
from all 7 FCV strains used in the present study. Maximum CT
(threshold cycle) value was obtained for F9 strain of FCV while the minimum
value was for NADC strain. The PCR products were separated on 1.5% agarose
gel and a single band at 151 bp position was observed for all seven strains
(Fig. 1). The primer/probe set failed to amplify this
region in CHV and FPV.
When 20 field samples were tested, 8 were found to be positive for
FCV by the conventional virus isolation method. Of these, 7 were found
to be positive by rRT-PCR.
Amplification Efficiency of rRT-PCR
Amplification efficiency of the assay was determined by using serial
10 fold dilutions of viral RNA from FCV 255 (85 μg mL-1)
as the PCR template. The assay was found to detect viral RNA up to 10-7
dilution (8.5 pg mL-1) over the range of linearity with slope
value of -3.29 and R2 (coefficient of correlation) 0.993 (Fig.
2). When RNA extracts of serial 10 fold dilutions of FCV 255 (initial
titer 1.45x109 mL-1) were used as PCR template,
the assay was able to detect RNA from virus suspension diluted to the
equivalent of 0.01 TCID50.
||Agarose gel separation of amplification products of
different FCV strains after real time RT-PCR. Lane 1 and 10: Molecular
marker; Lane 2: Feline panleukopenia virus; Lane 3: FCV 255;
Lane 4: F9; Lane 5: Urbana; Lane 6: FCV 2280; Lane 7: NADC; Lane 8:
FCV KCD; Lane 9: FCV CFI
||Standard curve obtained with serial 10 fold dilutions
of FCV 255 RNA. Ct values are plotted against different dilutions
of viral RNA, when used as template for real time RT-PCR
As with many other RNA viruses, FCV exhibits considerable antigenic heterogeneity
during replication in its host. Amino acid substitutions have been shown
to occur between residues 426 and 458 of FCV capsid protein E region indicating
that serotypic determinants of FCV important for antigenic variation are
contained in this region (Kreutz et al., 1998). Thus it is important
to develop a reliable, inexpensive and rapid system for the detection
of all different FCV strains.
We describe the development of an rRT-PCR assay for rapid detection of
FCV with high sensitivity and specificity. The assay was able to detect
as little as 0.01 TCID50 of FCV. The sensitivity of nPCR protocol
described by Marsilio et al. (2005) was 1000 fold greater than
virus isolation in cell culture (nPCR was positive at 10-11
dilution of stock virus whereas virus isolation was positive to 10-8
dilution). However, nPCR requires the use of two sets of primers for specific
amplification followed by separation of PCR products on agarose gel thus
increasing time of detection and risk of contamination. In our protocol,
the rRT-PCR is completed within 3 h without any need for the analysis
of PCR products, thus reducing the time of diagnosis. Helps et al.
(2002) reported an rRT-PCR for detection of FCV by melting curve analysis
using SYBR green I dye which was more sensitive than conventional PCR
as this could detect wide range of field isolates. However, this method
is not as sensitive as TaqMan assay since SYBR green dye binds to any
double stranded DNA produced by reverse transcription resulting in non-specific
products (Helps et al., 2002). The set of probe/primers used in
this study did not detect CHV and FPV suggesting that the described rRT-PCR
protocol is specific for the detection of FCV.
The newly developed rRT-PCR was able to detect 7 of 8 field samples this
assay may not detect all FCV isolates. Helps et al. (2002) noted
that FCV isolates are highly variable and that no single probe is able
to detect all the isolates. Although a major step forward in the rapid
detection of FCV, we believe that studies need to be continued to develop
single unified tests that can detect all field strains of FCV. Since the
detection limit of rRT-PCR is 0.01 TCID50, this test should
be useful in detecting carrier animals that are known to shed small quantities
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