Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Abstract

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV-AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV-AP. The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV. In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Preparation of Polyclonal Antibodies to Grapevine fanleaf Virus Coat Protein Expressed in Escherichia coli

“…Like other virus systems such as Faba bean necrotic yellows virus (Kumari et al, 2001), sugarcane yellow leaf virus (Korimbocus et al, 2002), Potato mop-top virus (Cerovska et al, 2003(Cerovska et al, , 2006 and Groundnut bud necrosis virus (Jain et al, 2005) where tagged viral CP was expressed in insoluble fraction, the fusion GFLV CP was detected in the insoluble fraction (pellet) from the transformed E. coli. Expression of recombinant fusion protein in the soluble fraction has also been reported elsewhere (Hema et al, 2003;Hourani and Abou-Jawdah, 2003;Lee et al, 2008;Raikhy et al, 2007), but we detected only a small amount of the fusion protein in the soluble fraction.…”

Immunocapture and simple-direct-tube-reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Rice yellow mottle virus

“…The resulting cDNA was amplified in PCR with the RYMV conserved primers, antisense primer RYMV II and sense primer RYMV III at 100 µM (Fargette et al, 2002). Immunocapture RT-PCR technique has been utilized widely for detection of other plant viruses (Wetzel et al, 1992;Harper et al, 1999;Hema et al, 2003;Mansilla et al, 2003;Zein et al, 2008;Silva et al, 2011;Kiliç, and Yardimci;Sarovar et al, 2010;Mallik et al, 2012;Uehara-Ichiki et al, 2013). This technique has been reported to have higher sensitivity than Passive Haemagglutination Assay (PHA) and Latex Agglutination Reaction (LAR) and could detect the target virus in leaf extracts diluted from 10 −3 to 10 −5 (Uehara-Ichiki et al, 2013).…”

“…Immuno-capture-RT PCR is designed for highly specific detection of templates present in very low amounts (Wetzel et al, 1992 (Wetzel et al, 1992;Harper et al, 1999;Hema et al, 2003;Mansilla et al, 2003;Ling et al, 2007;Zein et al, 2008;Silva et al, 2011;Ha et al, 2012;Kiliç, and Yardimci;. Generally, immunocapture-RT-PCR protocol has been established for the virus sensitivity improvement in detection (Silva et al, 2011;Yang et al, 2012).…”

Molecular characterization of Indian isolate of peanut mottle virus and immunodiagnosis using bacterial expressed core capsid protein

“…Similar results of expression of recombinant His-tagged CP in the insoluble fraction have been reported for several plant viruses, pelargonium zonate spot virus [27], faba bean necrotic yellows virus [15], sugarcane yellow leaf virus [13], potato mop-top virus [5,6] and groundnut bud necrosis virus [12]. However, for some plant viruses such as sugarcane streak mosaic virus, cucurbit yellow stunting disorder virus, cymbidium mosaic virus, the His-tagged viral CP was expressed in soluble fraction [10,11,16,23]. The PAb against core CP successfully detected the expressed protein as well as the virus in infected peanut leaf samples.…”