Validation and standardization of designed N gene primer-based RT-PCR protocol for detecting Peste des Petits Ruminants virus in goats

Abstract

The Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) test is one of the most popular and specific diagnostic tests to easily recognize the Peste des Petits Ruminants virus (PPRV) genome in clinical samples. The sensitivity of the RT-PCR test depends on gene-targeted primer sets. The literature appears to be lacking in designing primers used in RT-PCR to detect PPRV genome in Bangladesh. This study aimed to develop an N gene based PPRV primer set, a standardized RT-PCR protocol, and its validity test by comparison with other available primers. A total of seventy clinical samples and ten PPRV positive isolates were used in real-time RT-PCR and conventional RT-PCR using one pair designed primer set NF/NR and three pairs of published gene F1/F2, F1b/F2b and N1/N2. N gene-based PPRV primer sets (NF/NR) were designed from a published sequence of PPRV (Accession number GQ122187.1). Statistical analysis was carried out. The designed N gene-based primer positive PPRV samples were sequenced and analysed. The N gene-based primer sets were more sensitive to PPRV detection than F gene-based primer (P =.002) in the RT-PCR test. PPRV detects the highest (86%) of clinical samples in the RT-PCR test using a designed N gene-based primer. Sequence analysis showed that designed N gene-based the 402bp sequence of PPRV isolates is clustered with other Bangladeshi PPRV isolates and belongs to Lineage IV. New primers sets were designed from the conserved region of the N gene of PPRV. Designed primer sets successfully worked in real-time RT-PCR. The standardized RT-PCR protocol with the designed primer sets (NF/NR) can be used for the specific detection of PPRV from clinical samples. [ J Adv Biotechnol Exp Ther 2022; 5(3.000): 497-509]

Keywords

• ppr virus
• design n gene primer
• standardize rt-pcr protocol
• comparison primer
• analysis