Immunocapture Polymerase Chain Reaction for the Detection and Characterization of Cacao Swollen Shoot Virus 1 A Isolates

Abstract

Oligonucleotides derived from the flanking regions of the putative coat protein gene of the cacao swollen shoot badnavirus isolate 1 A (CSSV-l A) were able to prime the synthesis of specific products directly from extracts from CSSV-l A-infected leaves by immunocapture polymerase chain reaction (IC-PCR), following trapping of virions with polyclonal antibodies to CSSV-l A. CSSV isolates serologically distinct from CSSV-l A were not detected by IC-PCR when the CSSV-l A-derived primers were used following trapping with homologous antisera. IC-PCR was at least 100-fold more sensitive than double antibody sandwich (DAS)-ELISA in comparative tests on samples from greenhouse-grown cacao plants. The superior sensitivity of IC-PCR over DAS-ELISA was confirmed in attempts to detect and identify CSSV-l A isolates in field samples and permitted detection of CSSV-l A isolates even in symptomless leaves from plants showing stem swelling only. The IC-PCR products obtained from four randomly selected field samples were sequenced and shown to contain a region of the CSSV-1 A genome where ORF X overlaps ORF 3. Analysis of the partial amino acid sequences deduced from ORF 3 and ORF X of the four field isolates revealed a considerable variation in these CSSV-l A gene products.

Early signs of infection in Cacao swollen shoot virus (CSSV) inoculated cocoa seeds and the discovery of the cotyledons of the resultant plants as rich sources of CSSV

“…A more extensive studies between cotyledons and leaves of infected plants presented in Table 3 also showed the cotyledons to be a better source of CSSV: Out of 23 PCR analysis carried out on 23 total DNAS extracted using Dneasy Plant Mini Kit (Qiagen) from the two types of tissues (that is cotyledons or leaves) from five batches of infected plants planted at different times, 83% were CSSV positive, that is 83% (19/23) of CSSV DNA was amplified from the cotyledons as compared to the 9% (2/23) that was amplified from the leaf extracts. The results put together highlight the difficulty involved in working with cocoa leaves, thus corroborating previous reports Dzahini-Obiatey and Ollennu, 2000;Hagen et al, 1994;Hoffmann et al, 1997;Jacquot et al, 1999;Muller et al, 2001;Sagemann et al, 1983Sagemann et al, , 1985. It was generally easier to extract total DNA by Qiagen's DNeasy Plant Mini Kit system from the cotyledons than from the cocoa leaves.…”

“…By showing that CSSV could be amplified or detected in inoculated seeds as young as 3 dpi, coupled with the proof that PCR amplification of CSSV from the cotyledons are more efficient and reliable than from cocoa leaves, presents yet another opportunity for developing rapid diagnostic tools for CSSV detection or screening, which can then be used in CSSV-resistant breeding programmes for cocoa. It is also noteworthy that, there was no need to increase the number of PCR cycles for the virus to be amplified from the different isolates of CSSV studied in this project as against what was reported by Muller et al (2001), nor was it necessary to further process the CSSV DNA prior to amplification as was carried out by Hoffmann et al (1997). An added advantage of working with cotyledons was that, total DNAs were easier to extract from them than was the case for the leaves.…”

“…There was little or no mucilage to grapple with, and pipetting was easier. Judging from the ease with which CSSV DNA was regularly extracted and amplified from the cotyledons in this study, as compared to the problems others faced working on cocoa leaves (Hagen et al, 1994;Hoffmann et al, 1997;Jacquot et al, 1999;Muller et al, 2001;Sagemann et al, 1983Sagemann et al, , 1985. It could then be said that mucilage may be the culprit in cocoa leaves, which could have interfered with biochemical and molecular analysis previously reported (Hagen et al, 1994;Hoffmann et al, 1997;Jacquot et al, 1999;Muller et al, 2001;Sagemann et al, 1983Sagemann et al, , 1985.…”

“…For many years now, attempts to develop rapid diagnostic methods for screening CSSV-resistant cocoa cultivars during breeding programmes have been elusive; hence the painfully slow pace of breeding for CSSV-resistant cocoa in West Africa. The cocoa leaves, the only tissue, which has so far been tried for the development of previous diagnostic assays, is fraught with numerous problems Dzahini-Obiatey and Ollennu, 2000;Hoffmann et al, 1997;Jacquot et al, 1999;Muller et al, 2001;Sagemann et al, 1983Sagemann et al, , 1985, notable among which are the fact that the leaves contain inhibitors of PCR (Hoffman et al, 1997;Muller et al, 2001;Sackey et al, 1995Sackey et al, , 1996Sackey et al, , 1999 immunological (Hoffmann et al, 1997;Sagemann et al, 1983Sagemann et al, , 1985 and other molecular and biochemical assays DzahiniObiatey and Ollennu, 2000;Sackey et al, 1999). This is why the discernment of cellular-modifications by microscopy, which is reported in this study, is so important.…”

“…Lanes are: CSSV 1A (1-4), CSSV Bisa (5-8), CSSV N1 (9-12), CSSV Kpeve (13-16), un-inoculated (healthy) control (17-19) and Bioline's HyperLadder IV (20). molecular, biochemical or serological studies (Hagen et al, 1993(Hagen et al, , 1994Hoffmann et al, 1997;Jacquot et al, 1996Jacquot et al, , 1999Muller et al, 2001) and the cotyledons, which have just been found to contain CSSV in extractable quantities. The results which have been presented in Figure 5 and Table 3 showed that the cotyledons could be a better source of CSSV than the leaves.…”