Spot-test identification and rapid quantitative sequential analysis of dipyrone

A qualitative spot-test and tandem quantitative analysis of dipyrone in the bulk drug and in pharmaceutical preparations is proposed. The formation of a reddish-violet color indicates a positive result. In sequence a quantitative procedure can be performed in the same flask. The quantitative results obtained were statistically compared with those obtained with the method indicated by the Brazilian Pharmacopoeia, using the Student's t and the F tests. Considering the concentration in a 100 µL aliquot, the qualitative visual limit of detection is about 5×10-6 g; instrumental LOD ≅ 1.4×10-4 mol L-1 ; LOQ ≅ 4.5×10-4 mol L-1.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Identification and characterization of a Hydrangea ringspot virus isolate infecting hydrangea in São Paulo State

Hydrangea plants showing leaves with chlorotic and necrotic rings from Arujá Municipality, São Paulo State, were analyzed for the identification of the viral species. Elongated filamentous particles of 490 nm were visualized under transmission electron microscope. Oligonucleotides for Hydrangea ringspot virus (HdRSV), a potexvirus commonly found in Europe and in the United States, were tested using total RNA from hydrangea plants, amplifying two fragments, one around 550 and another one of 250 nucleotides. Nucleotide identity with HdRSV (accession number AJ 707100.1) was 96% and 88% for the longest and shortest fragment, respectively, indicating the presence of this virus. To evaluate its dissemination in the matrices of hydrangea used in the commercial production, 17 samples were collected in the region of Arujá, and eight were infected by HdRSV. For the analyzed viral replicase portion, the isolates from the varieties 'Azul LZR', 'Rosita', 'Renat Blue' and 'Vermelho Comum' did not differ in their amino acid sequences from isolates with sequences deposited in the GenBank (accession numbers AY 707100 and NC_006943). The isolates from 'Azul Rendado' and "Rosa Japonesa' showed few differences but were related to the remaining isolates. An antiserum was obtained for HdRSV and can be efficiently used to detect such virus in hydrangea and Primula malacoides, another ornamental plant also infected by HdRSV.