Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Evaluation of protective efficacy of CC-2 formulation against topical lethal dose of T-2 toxin in mice.

T-2 toxin is the type-A trichothecene and a common contaminant of food and cereals, produced by Fusarium species. T-2 toxin easily penetrates skin due to its lipophilic nature and causes skin irritation and blisters in humans. Physical protection of the skin and airway is the only proven effective method of protection. To date, no chemical antidotes are available to prevent T-2 induced lethality. In the present study, we evaluated the protective efficacy of 20% N,N'-dichloro-bis(2,4,6-trichlorophenyl) urea (CC-2) formulation against lethal topical exposure dose of T-2 toxin in mice. None of the animals exposed to only T-2 toxin at lethal dose of 2 and 4 LD50 (11.8 and 23.76 mg/kg body weight) survived beyond 36 and 16 h, respectively. CC-2 application at 5 and 15 min post-exposure protected mice 100% from lethality at 2 LD50. Survival rate was 100% and 50% at 4LD50 dose if CC-2 was applied dermally within 5 and 15 min post-exposure. Recovery profile of surviving animals after 2LD50 T-2 toxin exposure at 1, 3, 7, and 14 days was assessed in terms of hepatic GSH, lipid peroxidation, serum ALP, ALT and AST. Hepatic lipid peroxidation significantly increased in all groups exposed to T-2 toxin by 3 day but normalized by day 7. A delayed GSH depletion was noted in surviving animals on day 7 but recovered by day 14. ALT and AST levels were elevated in all CC-2 protected mice on day 1 and normalized by day 3. ALP level decreased till day 7 in all protected groups. The biochemical variables recovered to control values by 14th day. GC-MS analysis after in vitro interaction of CC-2 formulation with T-2 toxin had shown that nearly 86% of T-2 toxin is decontaminated in 5 min but 8-10% of T-2 toxin was still present even after 60 min of interaction. Results of our study suggest that CC-2 may be an effective dermal decontaminant against lethal topical exposure of T-2 toxin.

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L.

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2-6%) of sodium alginate and complexed with calcium chloride (25-100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0-49.7% in vitro and 2.0-4.5% in vivo.