Production of polyclonal antibodies to the coat protein gene of Indian isolate of Apple stem grooving virus expressed through heterologous expression and its use in immunodiagnosis (original) (raw)
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Phytoparasitica, 2018
Majority of the apple trees are known to be infected by two latent viruses, Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV). The importance of ASGV and ASPV is due to their non expression of symptoms, worldwide occurrence and wide host range on pome and stone fruits. Due to their latent nature in apple, early and rapid diagnostics plays important role for production of virus free quality planting material. The present investigation was conducted to detect and quantify ASPV & ASGV from different plant parts (spatial) in apple trees during different seasons (temporal) for optimisation of tissue and time for their rapid and early detection. Detection and relative quantification using immuno-molecular diagnostic techniques like, Double Antibody Sandwich-ELISA, Reverse Transcription-PCR and Real Time RT-PCR in various plant parts (leaf, whole flower, sepal, petal, anther, stigma with style, bark, fruit, seed and root) during different seasons was done. The DAS-ELISA based detection revealed infection in all plant parts except root and fruit with ASGV and ASPV, showing more expression in leaves followed by bark and whole flower. Similar results were also observed on RT-PCR based detection. Quantitative real time PCR analysis showed variation in expression of ASGV and ASPV in different parts during different seasons. Results confirmed that the ASGV and ASPV expression is higher in leaves followed by bark and whole flower. Periodic detection of these viruses in different plant parts during all the four seasons revealed varied virus titer from one season to another in the same plant. During all the seasons, both ASPV and ASGV were detected in bark in measurable titer using immunomolecular detection tools, however via DAS-ELISA, ASGV remained undetected during dormant season. Hence leaves and bark except leaf during fall, can be directly used as detection material for their early and rapid detection leading to production of virus free planting material.
The Plant Pathology Journal
Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcriptionrecombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as 4.7 ng/μl of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of 42°C. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.
Journal of Plant Biochemistry and Biotechnology, 2011
Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.
Biotechnology(Faisalabad), 2015
Full length GFLV Coat Protein (CP) gene of Grapevine fanleaf Virus (GFLV) as one of the most destructive viruses of grapevine around the world was amplified from a previously prepared clone Kh4-5-3 with a pair of newly-designed primers. The CP gene was inserted into bacterial expression vector pET-21a (+) and the construct (pET21aGFLV CP) was cloned in Escherichia coli strain Rosetta. Optimization of expression was done by induction with 0.25, 0.5, 1, 2 and 6 mM final concentration of IPTG each for 3, 4, 6 and 16 h. Induction with 1 mM IPTG for 4 h proved to be the most efficient. Expression of the CP was verified with SDS-PAGE, Western blotting and Dot Immunobinding Blot Assay (DIBA) by the use of commercially available anti-GFLV antibody. The expressed CP was purified as the denatured or native protein before using as the antigen for raising anti-GFLV CP antiserum in rabbits. Specificity and titration of the antiserum was determined by Plate-Trapped Antigen Enzyme-Linked Immune Sorbent Assay (PTA-ELISA). Efficiency of the anti-GFLV CP IgG purified from the antiserum was demonstrated in Western blotting and Double Antibody Sandwich (DAS)-ELISA. This is the first report on preparation of polyclonal antibodies against recombinant GFLV CP isolate from Iran and its application in the virus detection.
Tropical Plant Pathology, 2013
Cowpea mild mottle virus (CpMMV), the causal agent of stem necrosis disease, has drawn attention of soybean producers in recent years due to yield losses in the main producing regions of Brazil. Serological methods for viral detection require the use of an antiserum of good quality to achieve specificity and sensitivity. The entire coat protein gene of a Brazilian CpMMV isolate was cloned into a bacterial expression vector and transformed into Escherichia coli BL21::DE3 for in vitro expression. The coat protein, fused to a His-tag, was purified under denaturing conditions by affinity chromatography using a Ni-NTA resin. After renaturation, the integrity and identity of the purified recombinant protein was confirmed by SDS-Page and MALDI-ToF/ToF mass spectrometer analyses. A rabbit was immunized with increasing amounts of the recombinant protein. The specificity and sensitivity of the antiserum was demonstrated by Western blot and indirect ELISA assays. The polyclonal antisera raised against recombinant coat protein proved to be a reliable tool for CpMMV detection.
Letters in Applied Microbiology, 2021
The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the p...
Determination of the effects of Apple stem grooving virus on some commercial apple cultivars
Studies to determine the effects of Apple stem grooving Capillovirus (ASGV) on external and physical characteristic of some commercial apple (Malus dometsica Borkh.) cultivars were carried out in the Adana Plant Protection Research Institute's screen house facility in Turkey during 2006-2008. The selected cultivars for this aim were 'Jersey Mac', 'Fuji', 'Golden Delicious', 'Summer Red', 'Granny Smith', 'Vista Bella', 'Galaxy Gala' and 'Starking'. The selection of the cultivars was based on their common use by growers in the country. All cultivars were grafted on M9 rootstock and potted in the screen house. Turkish io-50 ASGV isolate, which had been obtained from previous works from an 'Anna' apple tree, was used for inoculation by chip budding, and the success of inoculation was confirmed by DAS-ELISA. The trial was evaluated two years after inoculation, based on six external and two physical parameters of ...
Simultaneous Detection of Major Pome Fruit Viruses and a Viroid
Indian Journal of Microbiology, 2013
A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of *198, *330, *370, *547 and *645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.
Simple and Quantitative Detection of Apple latent spherical virus Vector by a Spot Hybridization
Apple latent spherical virus (ALSV) vector has been widely used as a vector for virus-induced gene silencing, gene expression, and vaccination for protection from virus diseases in wide variety of plants. Here we describe a simple and quantitative method for ALSV detection in plant materials. ALSV infected plant leaves were homogenized in a buffer, and added with NaOH, which was found to be optimal at the final concentration of 10 mM. After serial dilution with 10 mM NaOH, the samples were spotted onto nylon membrane. After neutralization with acidic solution and fixation of nucleic acid by UV irradiation, the membrane was subjected to a prehybridization for an hour, followed by a hybridization with digoxygenin-labelled RNA probe for overnight. ALSV was quantitatively detected using a chemiluminescent substrate. The entire protocol took two days with hands-on time of a couple of hours.