Immunocapture-reverse Transcription-polymerase Chain Research Articles

Immunocapture reverse transcription polymerase chain reaction for detection of sugarcane streak mosaic virus

Detection of plant viruses can be done by protein or nucleic acid approaches. The immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) method is a combination of the two approaches. Research was carried out to develop and validate IC-RT- PCR based-detection method for SCSMV, which can be applied for the sugarcane seed indexing program to support the national government’s goal for sugar self-sufficiency. Evaluation of the IC-RT-PCR method was conducted using 5 field samples. Conventional PCR and serological methods, i.e. dot immunobinding assay (DIBA) and enzyme-linked immunosorbent assay (ELISA) was also performed in the same time. All field samples gave a positive reaction to SCSMV antibodies in the DIBA and ELISA methods with the intensity of the reaction varying from low to high. SCSMV was still detected on plant extract up to 104 dilution by ELISA and DIBA. Specific DNA fragments were successfully amplified from 2 field samples using the conventional PCR method; whereas the IC-RT-PCR method was successfully amplified all field samples. Optimization test showed that the IC-RT-PCR method was able to detect SCSMV from plant extract up to 10−10 dilutions. IC-RT-PCR method is more sensitive than conventional PCR and might be recommended for the indexing method to produce high-quality virus-free sugarcane seed.

A New Tymovirus Isolated From Solanum quitoense: Characterization and Prevalence in Two Solanaceous Crops in Ecuador.

Naranjilla (Solanum quitoense Lam.) and tamarillo (S. betaceum Cav.) are two important perennial solanaceous crops grown in Ecuador for the fresh market and juice production. Viruses infecting tamarillo and naranjilla are currently poorly studied, and no clean stock program exists in Ecuador. Here, we report a new virus, provisionally named as naranjilla mild mosaic virus (NarMMV) (genus Tymovirus, family Tymoviridae), isolated from naranjilla grown in an orchard in Pichincha Province, Ecuador. The complete genome of the virus consists of 6,348 nucleotides and encodes three open reading frames typical for members of the genus Tymovirus. Phylogenetically, Chiltepin yellow mosaic virus, Eggplant mosaic virus, and the recently characterized naranjilla chlorotic mosaic virus (NarCMV) were found to be the closest relatives of NarMMV. Unlike NarCMV, the new virus induced mild mosaic in naranjilla and more severe symptoms in tamarillo. Similar to NarCMV, NarMMV was unable to systemically infect potato. Virus surveys found NarMMV prevalent in naranjilla production areas of two provinces of Ecuador, especially where hybrid cultivars of naranjilla were cultivated. NarMMV was also found in field-grown tamarillo. The new virus cross-reacted with antibodies developed against NarCMV. Hence, this antibody will be useful for its field diagnosis using enzyme-linked immunosorbent assay or immunocapture reverse transcription polymerase chain reaction in future virus-free certification programs.

Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

Induced systemic resistance against Cucumber mosaic cucumovirus and promotion of cucumber growth by some plant growth-promoting rhizobacteria

During the investigation Cucumber mosaic cucumovirus (CMV) was isolated from cucumber plants showing virus like symptoms depending on indirect enzyme linked immunosorbant assay (I-ELISA) and Chenopodium quinoa as local lesion host. Three isolates from the predominant plant growth-promoting rhizobacteria (PGPR) were isolated from cucumber plants rhizosphere, and identified morphologically and physiologically to be related to Bacillus subtilis, Pseudomonas fluorescens and Azotobacter chroococcum species. The bacterial liquid crude cultures (72h of age) and their supernatants were tested for their ability to induce systemic resistance within cucumber plants (Cucumis sativus L. cultiver Beit Alpha) against CMV infection. Two types of treatment were carried out: (1) spraying of healthy cucumber plants (carrying 4–5 leaves) and challenging by mechanical CMV inoculation at time intervals (5–10days), (2) irrigation, as healthy cucumber seeds were irrigated with 200ml from each bacterial culture or their supernatants and inoculated with CMV 15days post treatment. Data proved that best results were obtained by treatment of irrigation with the Azotobacter crude culture, as the number of symptomless plants were 11 out of 30 plants inoculated, followed by Pseudomonas treated plants which gave eight asymptomatic plants. The induced resistance was tested using I-ELISA and immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) for the detection of CMV coat protein gene (cp), which proved that the mentioned symptomless plants were virus-free or with a low level of virus infection. Azotobacter treated plants giving virus-free results revealed the higher peroxidase and β-1,3-glucanase enzyme activities, 7U/gm and 500nktal/gm, respectively. Using gel electrophoresis and in comparison with control plants, a new protein band was detected in the protected cucumber plant extracts (molecular weight of about 30KDa), assuming to be a plant pathogen related protein. Increase in growth measures was observed for Azotobacter and Pseudomonas treated cucumber plants, as the higher plant dry weights were 16.1 and 13.8gm, respectively. Statistical lowest significant differences test (LSD) showed significant differences between Azotobacter and Pseudomonas results for biological data of plant dry weights.

Potato virus surveys and wide spread of recombinant PVYNTN variant in Central Tunisia

Surveys in the late season crops of Central Tunisia, one of the main potato growing areas, revealed the presence of the six most economically devastating viruses: potato leaf roll virus: PLRV: (Polerovirus), potato virus S (PVS) and M (PVM) (Carlavirus), potato virus X (PVX: Potexvirus), potato virus A (PVA) and Y (PVY) (Potyvirus) with large differences in incidence. Infection rates at harvest, assessed by serological test ranged from 0.5% (PVM) to 71% (PVY). The characterization of PVY variability was analyzed with a combination of serotyping, indexing on tobacco and reverse transcription polymerase chain reaction (RT-PCR) tests targeting 2 genomic regions (5’NTR/P1 and CP/3’NTR). Serological analysis of the collected samples revealed dominance of the PVYN group (88.2% of the total number of singly PVY positives). Furthermore, molecular typing of strains revealed that 73.3% of the PVYN group were typical PVYNTN variants with a recombination junction in CP/3’NTR region for 94.4% of them, whereas no recombination junction was identified in the genome of the isolates belonging to the PVYN group. To our knowledge, this is the first report of the occurrence of the PVYNTN variant and its high incidence in late season potato growing areas of central Tunisia. Key words: Diagnosis, immunocapture reverse transcription polymerase chain reaction (RT PCR), potato viruses, potato virus Y (PVY) diversity, Central Tunisia.

DEVELOPMENT OF PCR-BASED TECHNIQUES FOR THE DETECTION OF IMMOBILISED POTATO VIRUS Y VIRIONS

Detection of Potato virus Y (PVY), one of the important potato viruses, was undertaken using various PCRbased techniques, i.e immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR), direct binding RT-PCR (DB-RT-PCR) and print capture RTPCR (PC-RT-PCR). These techniques were evaluated for detection of PVY virions immobilized on solid supports. The 5’UTR and P1 region (969 bp) of the PVY genome were amplified using these techniques, which were compared with DAS-ELISA and nucleic acid spot hybridization (NASH) using fluorescein labeled cDNA probes. The detection limit of purified PVY in serially diluted healthy potato leaf sap was 10 ng for DAS-ELISA, 1 ng for NASH and DB-RT-PCR, 1pg for PC-RT-PCR, ICRT- PCR and standard RT-PCR. Of all techniques, PCRT- PCR proved to be the simplest, economical and reliable. It does not require antibodies for the capture of virions nor lengthy RNA extraction processes.

Immunocapture RT-PCR을 이용한 박과작물 종자전염 바이러스의 검출

Immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) was applied to the detection of Cucumber green mottle mosaic virus (CGMMV), Kyuri green mottle mosaic virus (KGMMV), and Zucchini green mottle mosaic virus (ZGMMV) on Cucurbitaceae crops. These seed-borne tobamoviruses were accurately detected from the infected leaves and seeds by IC-RT-PCR. This method was estimated to be about 100 times more sensitive than ELISA, and also it allowed the direct confirmation of ELISA results by using the captured antigens from a completed ELISA microwell. This convenient and reliable method could be used routinely for large-scale field surveys or seed tests of Cucurbitaceae crops.

Detection of potato leafroll virus isolated from potato fields in Tehran province in aphids by immunocapture reverse transcription polymerase chain reaction

The surveys conducted during 2006 and 2007 revealed the infection of the virus in potato fields in Tehran province. Due to the important roll of aphids in transmission of the virus, immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) and reverse transcription polymerase chain reaction (RT-PCR) was developed using potato leafroll virus (PLRV) specific antibodies and specific primer pair (20 mer) located in the virus capsid gene to detect the virus in aphids. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined in PLRV-infected plants. This inquiry shows that this method is applicable to assess viroliferous nature of aphids in yellow -pan traps during season.

Detection and identification of the blackeye cowpea mosaic strain of Bean common mosaic virus in seeds of cowpea from southern India

In different legume-growing regions of India, a total of 136 seed samples of cowpea (Vigna unguiculata (L.) Walp.) were collected and tested for the presence of the blackeye cowpea mosaic strain of Bean common mosaic virus, Cowpea aphid-borne mosaic virus, Cowpea mosaic virus, Cucumber mosaic virus and Bean yellow mosaic virus using growing-on test, enzyme linked immunosorbent assay, differential host test, electron microscopy and immuno-capture reverse transcription polymerase chain reaction. Among the 136 seedlots tested, 43 cowpea seedlots were found to be infected with BCMV-BlCM. The identity of three of these isolates as BCMV-BlCM was further supported by nucleotide sequencing in the 3′ region of the genome. The incidence of seeds carrying transmissible virus ranged from 0.67% to 13.49%. In most cases, only symptomatic seedlings in a growing-on test were found infected with the virus.

Cross-Protection as Control Strategy Against Grapevine fanleaf virus in Naturally Infected Vineyards.

The efficacy of cross-protection at mitigating the impact of Grapevine fanleaf virus (GFLV) on grapevines (Vitis vinifera) was assessed in two naturally infected vineyard sites. Test vines consisted of scions grafted onto rootstocks that were healthy or infected by mild protective strains GFLV-GHu or Arabis mosaic virus (ArMV)-Ta. Challenge GFLV infection via the nematode Xiphinema index was monitored over nine consecutive years in control and ArMV-Ta cross-protected vines by double-antibody sandwich-enzyme-linked immunosorbent assay using GFLV-specific antibodies, and in GFLV-GHu cross-protected vines by characterizing the coat protein gene of superinfecting isolates by immunocapture-reverse transcription-polymerase chain reaction-restriction fragment length polymorphism. Results were consistent with a significantly reduced challenge infection rate in cross-protected vines compared with control vines, more so in those protected with GFLV-GHu (19 versus 90%) than with ArMV-Ta (40 versus 65% in field A and 63 versus 90% in field B). However, the two mild strains significantly reduced fruit yield by 9% (ArMV-Ta) and 17% (GFLV-GHu) over 8 years and had a limited effect on fruit quality. Therefore, in spite of a great potential at reducing the incidence of challenge field isolates, cross-protection with natural mild protective strains GFLV-GHu and ArMV-Ta is not attractive to control GFLV because the negative impact on yield is a limiting factor for its deployment.

Cloning and sequence analysis of coat protein gene for characterization of sugarcane mosaic virus isolated from sugarcane and maize in Thailand

Three isolates of sugarcane mosaic virus (SCMV) from sugarcane and maize in Thailand, were selected and used as viral sources for coat protein (CP) genes cloning by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR). The CP gene sequences and their deduced amino acids were determined. The sequences of three cloned CP genes, UT6TH-sgc and UD7TH-sgc from sugarcane, and SBC2TH-mz from maize were compared with other previously reported sequences of SCMV-CP gene and their phylogeny were studied. The analysis revealed that Thai SCMV CP gene contained 942 nucleotides encoded for coat protein MW of 33.65 kDa. The nucleotide sequence identity among cloned CPs from three isolates were 98-99% to each other. The N-terminus of the Thai SCMV CP contained a distinctive sequence, especially at nucleotide positions 28-46 of the N-terminal variable 70 amino acid residues which discriminated Thai SCMV from all previously reported SCMV isolates. Thai SCMV were placed in a separate branch of SCMV-MDB cluster which is closely related to most of SCMV isolates from maize, and distinct from the other cluster containing sugarcane-SCMV strains.

Phytosanitary status of potato in the Bekaa valley in Lebanon

The incidence, severity and distribution of four virus diseases of potato were determined in the main production areas of Lebanon during the 2001 and 2002 growing seasons. Observations were also recorded on diseases incited by fungi and bacteria, as well as physiological disorders. The surveys covered 40 randomly selected potato fields from three different areas of the Bekaa valley, the main potato production region of Lebanon. 715 samples were individually collected and tested by DAS‐ELISA for Potato virus A (PVA), Potato virus X (PVX), Potato virus Y (PVY) and Potato leafroll virus (PLRV). 51% of the total tested samples were infected by one or more viruses. PVY was the most prevalent virus, detected in 98.8% of the total number of infected samples, followed by PVA and PVX (both at 10.6%) and PLRV (9.3%). Mixed infections were about 17%. Potato tuber necrotic ring spot disease, caused by PVYNTN, was determined by immunocapture reverse transcription polymerase chain reaction. The fungi Thanatephorus cucumeris, Verticillium dahliae, Fusarium sp., Sclerotinia sclerotiorum and the bacterium Erwinia carotovora were the main pathogens found to be associated with the diseased plants. Quarantine pests such as Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum were not encountered. Growth cracks, hollow heart and internal rust spot were the predominant physiological disorders observed.

Detection of cherry leaf roll nepovirus (CLRV) in birch, beech and petunia by immuno‐capture RT‐PCR using a conserved primer pair

SummaryViruses in woody forest plants are known to be difficult to detect owing to phenolic compounds in plant extracts and an often irregular distribution of the pathogens within the plants. We describe a modified immunocapture reverse transcription polymerase chain reaction (RT‐PCR) to detect cherry leaf roll virus (CLRV) in forest trees. The application of a conserved primer pair is shown to be suitable for the detection of different isolates of CLRV from birch from distant forest stands in Germany and also in strains of CLRV from other host plants, including beech, walnut and petunia hybrids. The CLRV isolates and strains could be distinguished by direct sequencing of their PCR products. CLRV was easily detectable throughout the year in tissue of male inflorescences, leaf buds, leaves, single seeds, cortical tissues of young twigs and also in single aphids and bugs (Kleidocerys resedae) collected from a CLRV‐infected birch‐ tree.

Single-step immunocapture RT-PCR in the detection of raspberry bushy dwarf virus.

An immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) method for a highly sensitive analysis of raspberry bushy dwarf virus (RBDV) in infected plants is described. In the method, preliminary purification of virus particles or viral RNA from the plant material is not necessary. Viruses are enriched during the assay by antibodies bound in the PCR microplate wells, followed by lysis of the viral particles, and RT-PCR of the viral RNA. The reaction mixtures, including reverse transcriptase and DNA polymerase, have been selected so that both enzymes are active in the lysis and amplification conditions; by this way, it is possible to conduct the whole procedure in a single step. Using the method, four fragments from RNA-3 of RBDV have been amplified with various combinations of four primers. The procedure is sensitive enough to allow a simple detection of RBDV in in vitro cultured plants in which the detection of viruses by conventional immunological methods is difficult or even impossible.