Development of Sybr Green 1 Based Real-Time Polymerase Chain Reaction for Detection and Differentiation of Infectious Bursal Disease Virus
The current available method to differentiate very virulent and vaccine strains of infectious bursa1 disease virus (IBDV) is by restriction fragment length polymorphism of VP2 gene. However, this method is time consuming, errorproned and less sensitive. The newly developed TaqMan real-time PCR is...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2005
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| Online Access: | http://psasir.upm.edu.my/id/eprint/6605/1/FPV_2005_5.pdf http://psasir.upm.edu.my/id/eprint/6605/ |
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| Summary: | The current available method to differentiate very virulent and vaccine strains
of infectious bursa1 disease virus (IBDV) is by restriction fragment length
polymorphism of VP2 gene. However, this method is time consuming, errorproned
and less sensitive. The newly developed TaqMan real-time PCR is
very sensitive but not suitable for routine test as it is expensive. Additionally,
the application of the assay in detecting very virulent and vaccine strains of
IBDV has not been reported. In this study the performances of SBYR Green
1 real-time, ELlSA and conventional agarose detection methods in detecting
nested PCR products were compared. It was found that the real-time PCR
was at least 100 times more sensitive than ELlSA detection method with a
detection limit of 250 pglpl. The developed assay detects both very virulent
and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. However, the assay was unable
to differentiate the different strains of IBDV. In the subsequent studies,
strain-specific primer (match primer) combinations were used for the
detection and differentiation of IBDV strains using two steps SYBR Green 1
based real-time PCR. The primers and PCR condition were optimized and
validated using both very virulent and vaccine strains. By using the strainspecific
primer combinations, specific amplification based on measurement of
CT and Tm were detected. In an optimized PCR condition, specific
amplification associated with early amplification with CT value between 19 to
28 and Tm between 86 to 88OC meanwhile nonspecific amplification from
mismatch primer was associated with late amplification with CT value > 29
and Tm < 82OC or no amplification (CT value 0 and Tm < 82OC). These
characteristic CT and Tm values were consistently detected following
amplification with 4000 nglul of cDNA. Hence, the differentiation of IBDV
strains was based on the detection of CT values whilst detection of Tm was
for confirmation of the specific amplification. The detection of Tm value alone
was not sufficient to differentiate IBDV strains. Even though the detection
limit of the real-time PCR to detect IBDV strains was between 6.6 to 7.7
nglpl, it is recommended that for testing of clinical samples, the cDNA
concentrations be maintained between 4000 nglpl to 66 nglpl for PCR
amplification, since amplification from insufficient primer-template
concentration promote amplification of mismatch PCR product. In this study,
it showed for the first time application of SYBR Green 1 based real-time PCR
for the detection and differentiation of very virulent and vaccine strains of IBDV. The assay was found to be sensitive, specific, less expensive and has
less turn around time compared to the current available diagnostic methods |
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