Duplex-immunocapture-RT-PCR for detection and discrimination of two distinct potyviruses naturally infecting sugarcane (Saccharum spp. hybrid).

A sensitive duplex-immunocapture-RT-PCR (D-IC-RT-PCR) technique was developed for detection and discrimination of taxonomically distinct Sugarcane streak mosaic virus (SCSMV) and Sugarcane mosaic virus (SCMV) that naturally infect sugarcane. D-IC-RT-PCR was performed using polyclonal antisera for capture of virions. Oligo 5′-d(T)18(AGC)-3′ as a common reverse primer for both viruses and virus specific forward primers, 5′-aagtggttaaacgcctgtgg-3′ and 5′-ATGTC(GA)AAGAA(GA)ATGCGCTTGC-3′ were used for amplifying ~1400 and ~900 bp fragments of SCSMV and SCMV genomes, respectively from their 3′ termini. To assess the applicability of the developed technique, 67 mosaic affected sugarcane samples were initially screened by direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) followed by D-IC-RT-PCR. In DAC-ELISA, ~69 % of tested samples were shown to be positive for presence of SCSMV, ~28 % for SCMV and ~10 % for both viruses. In D-IC-RT-PCR both viruses were detected up to the dilution of 10-4. In D-IC-RT-PCR, ~76 % of tested samples were found to be positive for SCSMV, ~37 % for SCMV and ~16 % for both viruses. The sequence analyses of D-IC-RT-PCR amplicons of 3 isolates of each virus revealed that the designed primers were virus-specific. The developed technique had potential application for sensitive parallel detection of two viruses in sugarcane.

Assembly of recombinant coat protein of sugarcane streak mosaic virus into potyvirus-like particles.

Coat protein (CP) gene of sugarcane streak mosaic virus-AP isolate (SCSMV-AP) was expressed in E. coli and recombinant CP (SCSMV-AP rCP) was purified by linear sucrose density gradient centrifugation. Observation of purified SCSMV-AP rCP under electron microscope revealed the presence of potyvirus-like particles (PVLPs). The assembled particles were shown to encapsidate CP gene transcripts by slot-blot hybridization.

Plasma cortisol response to 1-24 adrenocorticotropin in patients with treated/untreated sellar & suprasellar mass lesions.

BACKGROUND & OBJECTIVES: One microgram short synacthene test is widely recommended as a screening test for evaluation of hypothalamo-pituitary-adrenocortical axis in patients with secondary adrenal insufficiency. Information on adequacy of cortisol response to this dose at different periods of the day in patients with hypothalamic-pituitary disorders is not available. Hence, this study was designed to assess the adequacy of cortisol response to 1 microg 1-24 adrenocorticotropin (ACTH) at 0800 h and 1600 h in patients with sellar and suprasellar mass lesions. METHODS: Thirty five consecutive patients with sellar and suprasellar mass lesions with mean age of 43.0+/-14.4 yr and 36 healthy controls with mean age of 32.3+/-9.0 yr were studied after obtaining informed consent. Maintenance doses of glucocorticoids in these patients were discontinued appropriately. On day 1, prestimulated and stimulated plasma cortisol samples at 0800 h and at 30 and 60 min following i.v. bolus of 1 microg 1-24 ACTH were collected. While on day 3, plasma cortisol samples were similarly collected at 1600 h. Cortisol estimation was done by a sensitive and specific radioimmunoassay. Stimulated plasma cortisol of 500 nmol/l or higher was defined as a normal response. RESULTS: In healthy controls, the prestimulated and peak cortisol levels at 0800 h (377.5+/-93.3 and 729.1+/-183.2 nmol/l) were higher (P<0.001 and P<0.01) than those at 1600 h (230.1+/-75.7 and 665.8+/-138.6 nmol/l). All subjects had a cortisol response of 500 nmol/l or higher in response to 1 microg 1-24 ACTH both at 0800 and 1600 h. In the patients' group, the prestimulated plasma cortisol at 0800 h (250.3+/-169.7 nmol/l) was higher (P<0.001) than that at 1600 h (166.3+/-128.9 nmol/l), while the peak cortisol response was comparable (P>0.05) in the morning as well as in the evening (490.9+/-309.4 vs 464.8+/-318.4). In 27 patients (77%) the morning and evening stimulated cortisol response to 1 microg 1-24 ACTH was consistent (normal in 13 and subnormal in 14) but was discrepant in the remaining 8 (23%). In 7 of these 8 patients, cortisol response was normal at 0800 h but not at 1600 h, while in only one, normal response was seen at 1600 h but not at 0800 h. INTERPRETATION & CONCLUSION: The demonstration of normal peak cortisol response to 1 microg 1-24 ACTH at 0800 h but not at 1600 h in substantial number of patients with sellar and suprasellar mass lesions suggests preference to morning for performing this test.