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|Title:||Quantification of classical swine fever virus load by one-step TaqMan real-time RT-PCR assay|
Barman, Nagendra Nath
Das, S. K.
|Keywords:||Classical swine fever virus;Real time PCR;TaqMan probe;qRT-PCR|
|Abstract:||A fluorogenic-probe hydrolysis (TaqMan) real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay based on amplification of a 93 bp fragment from 5’un-translated region (5’UTR) of classical swine fever virus was used for detection and absolute quantification of the virus in clinical and tissue samples. For determining analytical sensitivity, an in vitro transcript RNA containing 5’UTR of classical swine fever virus (CSFV) strain Alfort187 from plasmid pCRXLV324-6 was used as a positive control and a standard for quantification of CSFV genomic RNA copies. The real-time quantitative RT-PCR (qRT-PCR) assay was used to assess the CSFV shedding from naturally infected pigs in whole blood, nasal swab and also in tissue samples. Used qRT-PCR was specific and sensitive as it could detect as low as 16.3 copies of CSFV genomic RNA. The assay was also reproducible as shown by satisfactory low intra-assay (0.80 % to 1.87 %) and inter-assay (1.00% to 3.80%) coefficient of variation with an efficiency of 102.3% and R2 of 0.993. Thus, the real-time qRT-PCR assay described here allows rapid, specific and sensitive laboratory detection and quantification of CSFV genomic RNA copies.|
|ISSN:||0975-0967 (Online); 0972-5849 (Print)|
|Appears in Collections:||IJBT Vol.18(4) [October 2019]|
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