Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 13
ONE STEP RT-PCR FOLLOWED BY MSSCP AS A NOVEL METHOD FOR A FAST AND RELIABLE
DETECTION AND DIFFERENTIATION OF MIXED INFECTION WITH NEWCASTLE DISEASE VIRUS OF
DIFFERENT ORIGIN.
L. Rabalski 1 , D. Nidzworski 1 , B. Szewczyk 1 , K. Śmietanka 2 , Z. Minta 2
1
Intercollegiate Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Department of Molecular Virology, Gdansk, Poland
2
National Veterinary Research Institute in Pulawy, Department of Poultry Diseases, Pulawy, Poland
MSSCP, differentiation of strands, RT-PCR, Newcastle disease
Introduction
Newcastle disease virus (NDV) is an avian enveloped virus
containing linear non-segmented single-stranded RNA (Lamb et
al., 2005). It belongs to the Mononegavirales order,
Paramyxoviridae family and Avulavirus genus. NDV strains are
classified into three major pathotypes: velogenic, mesogenic and
lentogenic (Beard and Hanson, 1984). Vaccination (with live
virus) does not protect against the spread of virulent form of the
virus. Detection of the field virus in immunized population of birds
by means of conventional diagnostic methods is costly and time
consuming. To overcome this disadvantage, we have applied
One Step RT-PCR and Multitemperature Single-Strand
Conformational Polymorphism (MSSCP) methods in our
laboratories to distinguish between pathogenic and vaccine
strains of the NDV. Classical single-strand conformational
polymorphism (SSCP) analysis is based on the observation that
single stranded DNA fragments attain a number of
conformational forms which may be separated by native
polyacrylamide gel electrophoresis giving a characteristic pattern
of electrophoretic bands. Point mutations, other minor changes in
nucleotide sequence, as well as physico-chemical conditions like
ionic strength, pH and temperature may have significant effect on
electrophoretic pattern of single stranded DNA (Orita et al.,
1989). By sequential changes of separation temperature it is
possible to increase the resolution of single-strand DNA bands;
this technique was named MSSCP (where M stands for
“multitemperature”) (Kaczanowski et al., 2001).
Materials & methods
SPF chickens housed in isolation were infected oculonasally with
three different strains of NDV (LaSota – lentogenic, Roakin –
mesogenic and Italy – velogenic). Swabs from cloaca and
oropharynx were collected at different time-points post infection
(Tab.1). After RNA isolation and One Step RT-PCR, 123bp DNA
of viral fusion (“F”) gene fragments were obtained (Fig. 1). PCR
products were denatured (3’ in 97C) and subjected to MSSCP
electrophoresis where, after silver staining, they gave specific
ssDNA band patterns.
Table 1: The experimental design
Actions
Inoculation of birds with
LaSota and Roakin NDV
strains
Inoculation of birds with Italy
NDV strain
Collection of cloacal and
oropharyngeal swabs
Day of experiment
1
3
3, 4 ,5 ,6 ,7, 10
Results
Three embryo-grown NDV strains (separately and mixed
together) were tested for the level of detection. We found it
possible to determine the presence of the minor variant of the
virus, even when its contribution was less than 0.1% of total
sample. After MSSCP analysis, characteristic ssDNA band
patterns were obtained for each NDV strain. Both artificially
mixed and swab samples showed a combined pattern of bands
making it easy to identify the composition of samples (Fig. 2).
Figure 1: 123bp PCR product at 7 days after infection (digit -
identification of the chicken, G - oropharyngeal swabs,
K – cloacal swabs, M - HyperLadder IV - 100bp-1000bp)
Figure 2: MSSCP patterns of 123bp PCR product (M -
HyperLadder IV - 100bp-1000bp, L – LaSota, R – Roakin,
I – Italy, number – level of matrix concentration)
Discussion & conclusions
MSSCP combined with One Step RT-PCR can be applied for the
detection and preliminary characterization of NDV without the
need for nucleotide sequencing. The ability to diagnose multistrain
infections and to detect the minor viral variant are the main
advantages of the proposed method. Additionally, we confirm the
observation that vaccination does not protect against virus
replication and shedding in chickens.
The developed method can be especially helpful in the detection
of field infection in flocks immunized with live vaccines provided
that we know the MSSCP pattern of an NDV strain used for
vaccination. In such cases an RT-PCR method alone yields
postitive results irrespective of the number of strains present in
the tested sample while the sequencing shows the overlapping
peaks of fluorsecence. If the MSSCP patterns of NDV present in
the sample are not known, there is a possibility to cut the bands
from the gel and sequence them separately.
References
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Detection of polymorphisms of human DNA by gel electrophoresis as
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2766-70.
2. Kaczanowski, R, Trzeciak, L, Kucharczyk, K (2001). Multitemperature
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3. Lamb, R, Collins,P, Kolakofsk, D, Melero J, Nagai,Y, Oldstone, M,
Pringle,C, Rima, B. Family paramyxoviridae. In: Fauquet, C, Mayo M,
Maniloff J, Desselberger, U, Ball, L. (Eds.) Virus Taxonomy, VIII th Report of
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