Anshul Sharma

A Nano-Au/C-MWCNT based label free amperometric immunosensor for the detection of capsicum chlorosis virus in bell pepper

Accurate and on time diagnosis of plant viruses is an essential prerequisite for efficient control in field conditions. A number of diagnostic methods have been reported with the required level of sensitivity. Here, we propose a label free immunosensor for efficient and sensitive detection of capsicum chlorosis virus (CaCV) in bell pepper. Antigen was immobilized over the surface of gold nanoparticle/multi-walled carbon nanotube (Nano-Au/C-MWCNT) screen printed electrodes using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross linking chemistry followed by interaction with groundnut bud necrosis virus (GBNV)/CaCV specific polyclonal antibody. The electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) using the redox indicator. Electrode surface characterization was done by performing scanning electron microscopy (SEM). Electrochemical studies showed positive results at different antigenic dilutions ranging from 10−2 – 8x10−5. The sensitivity of the immunosensor developed has been compared with direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) and the results showed that the immunosensor developed was 800-1000 times more sensitive, when compared to DAC-ELISA for CaCV detection. The immunosensor we have developed is economical and sensitive and could be used for immediate determination of the presence of virus in extracts from bell pepper leaves.

RAPD typing of Lactobacillus brevis isolated from various food products from Korea

In the present study, the fingerprinting technique, random amplified polymorphic DNA-PCR was evaluated to characterize 13 strains of Lactobacillus brevis, isolated from different vegetable products of South Korea. Two primers i.e. 239 and KAY3 were used. The primer 239 produced bands ranged from 500-4,000 bp and KAY3 primer produced bands with sizes from 600-4,000 bp. Both primers produced thirteen different RAPD profiles. Phylogenetic dendrogram showed that all the isolates could be divided into six major clusters both the primers. However, a few strains of L. brevis had similar profiles and were not well differentiated by RAPD.

Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly

Coat protein (CP) and RNA3 from Prunus necrotic ringspot virus (PNRSV-rose), the most prevalent virus infecting rose in India, were characterized and regions in the coat protein important for self-interaction, during dimer formation were identified. The sequence analysis of CP and partial RNA 3 revealed that the rose isolate of PNRSV in India belongs to PV-32 group of PNRSV isolates. Apart from the already established group specific features of PV-32 group member’s additional group-specific and host specific features were also identified. Presence of methionine at position 90 in the amino acid sequence alignment of PNRSV CP gene (belonging to PV-32 group) was identified as the specific conserved feature for the rose isolates of PNRSV. As protein–protein interaction plays a vital role in the infection process, an attempt was made to identify the portions of PNRSV CP responsible for self-interaction using yeast two-hybrid system. It was found (after analysis of the deletion clones) that the C-terminal region of PNRSV CP (amino acids 153–226) plays a vital role in this interaction during dimer formation. N-terminal of PNRSV CP is previously known to be involved in CP-RNA interactions, but our results also suggested that N-terminal of PNRSV CP represented by amino acids 1–77 also interacts with C-terminal (amino acids 153–226) in yeast two-hybrid system, suggesting its probable involvement in the CP–CP interaction.

DNA Profiling of Leuconostoc citreum Strains in Fermented Foods by Repetitive Element Polymerase Chain Reaction

To identify and discriminate the bacterial species at the subspecific level, rep-PCR is a reliable genomic fingerprinting tool. Fourteen strains of bacteria were isolated from different food sources, identified as Leuconostoc citreum using 16S rRNA gene sequencing, and amplified using rep-primers (REP, ERIC, and (GTG)5). Fingerprinting patterns generated bands in the range of 300-6,000 bp with REP, 150-6,000 bp with ERIC, and 200-1,700 bp with (GTG)5 primers. In UPGMA dendrogram analysis, 14 strains were clustered into three clades (I, II, and III) with all the primers, thus differentiating them at the molecular level. The present study revealed the differentiation of L. citreum strains using rep-PCR.

Molecular characterization of tospoviruses associated with ringspot disease in bell pepper from different districts of Himachal Pradesh

Bell pepper (Capsicum annuum L.), an important cash crop for the farmers of Himachal Pradesh was found to be affected with tospovirus like disease. An extensive survey was conducted in the bell pepper grown areas in the five districts of Himachal Pradesh to identify and characterize the causative agent. Hence, 60 symptomatic bell pepper plants exhibiting characteristics symptoms were collected from Solan, Sirmaur, Hamirpur, Kangra and Bilaspur districts. Out of 60 samples, 53 samples were found to be positive by DAS-ELISA with tospovirus group specific antiserum. To confirm the presence of tospovirus, DAC-ELISA was performed using GBNV/CaCV polyclonal antiserum and DAS-ELISA with two monoclonal antibodies i.e. TSWV, GRSV. All the 53 samples were found negative for TSWV and GRSV and positive for GBNV/CaCV. Further, eleven infected isolates from both poly-house and open field conditions were selected for characterization at molecular level. RT-PCR was performed with N gene specific primers for TSWV, GBNV and CaCV. The eleven samples selected for molecular identification were further found to be negative for TSWV and positive for CaCV using RT-PCR. One of the samples from district Sirmaur was found to be positive for mixed infection of GBNV and CaCV. N gene phylogenetic analysis of CaCV/GBNV provided important information about the movement and evolution of tospoviruses in Himachal Pradesh.

Molecular typing of Lactobacillus brevis isolates from Korean food using repetitive element-polymerase chain reaction

Lactobacillus brevis is a part of a large family of lactic acid bacteria that are present in cheese, sauerkraut, sourdough, silage, cow manure, feces, and the intestinal tract of humans and rats. It finds its use in food fermentation, and so is considered a “generally regarded as safe” organism. L. brevis strains are extensively used as probiotics and hence, there is a need for identifying and characterizing these strains. For identification and discrimination of the bacterial species at the subspecific level, repetitive element-polymerase chain reaction method is a reliable genomic fingerprinting tool. The objective of the present study was to characterize 13 strains of L. brevis isolated from various fermented foods using repetitive element-polymerase chain reaction. Repetitive element-polymerase chain reaction was performed using three primer sets, REP, Enterobacterial Repetitive Intergenic Consensus (ERIC), and (GTG)5, which produced different fingerprinting patterns that enable us to distinguish between the closely related strains. Fingerprinting patterns generated band range in between 150 and 5000 bp with REP, 200–7500 bp with ERIC, and 250–2000 bp with (GTG)5 primers, respectively. The Jaccard’s dissimilarity matrices were used to obtain dendrograms by the unweighted neighbor-joining method using genetic dissimilarities based on repetitive element-polymerase chain reaction fingerprinting data. Repetitive element-polymerase chain reaction proved to be a rapid and easy method that can produce reliable results in L. brevis species.

Analysis of Leuconostoc citreum strains using multilocus sequence typing

The objective of this study was to perform genetic diversity analysis of 13 strains isolated from South Korean foods by multilocus sequence typing (MLST). For typing, seven housekeeping loci (atpA, dnaA, dnaK, gyrB, pheS, pyrG, and rpoA) were selected, amplified and analyzed. Fifty-one polymorphic sites varying from 1 to 22 in each species were identified. Thirteen sequence types were generated with allele numbers ranged from 2 to 10. The overall relationship between strains was assessed by unweighted pair group method with arithmetic mean dendrogram and minimum spanning tree. In addition, combined spits tree analysis revealed intragenic recombination. No clear relationship was observed between the isolation sources and strains. The developed MLST scheme enhanced our knowledge of the population diversity of Leu. citreum strains and will be used further for the selection of industrially important strain.

DNA profiling of Leuconostoc mesenteroides strains isolated from fermented foods and farm produce in Korea by repetitive-element PCR

Lactic acid bacteria are known for their preservative effects on food products like meat and sausage. Since they are related to humans, these bacteria require proper characterization and identification among various other bacteria in the surroundings. For their identification, several typing methods have already been applied of which the genotyping methods provide reproducible and unambiguous results. In this study, PCR-based method called repetitive element PCR was used for typing 37 Leuconostoc mesenteroides with three primers, REP, ERIC, and (GTG)5, annealing to repetitive sequences present in the bacterial genome. Different fingerprints were obtained for the isolates showing distinguishing profiles. Further phylogenetic analysis was performed using UPGMA method of clustering which provided proper identification with genetic relatedness of all the isolates. It was finally observed that, out of the three primers used, (GTG)5 discriminated the strains precisely than the other two.