Molecular characterisation of Aspergillus flavus isolates from peanut fields in India using AFLP

Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the ‘A’, ‘B’ and ‘G’ group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84%) was more than that between two populations (16%). The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.

Smears from rat liver suspension as modified substrate for antinuclear antibody test.

Immunofluorescence still remains a standard method for documenting antinuclear antibodies (ANA). Cryostat cut sections of rodent liver or Hep2 cell nuclei have been used as substrate in the test but are often difficult to arrange in laboratories in developing countries. Hence, a modification was developed using smears from rat liver suspensions. The smears were compared with the cryostat cut sections over 338 sera samples of suspected cases of collagen diseases, rheumatoid arthritis, thyroid disorders, hepatitis B, enteric fever, tuberculosis and normal subjects. The sera from suspected collagen diseases cases were also compared with ANA test using Hep2 cells. The modified smear technique was well comparable and the clarity of the immunofluorescence was even better than for cryostat cut sections. Using the modified smear technique 272 sera out of 2,851 sera gave positive test for ANA. The homogenous, speckled and peripheral patterns were seen for 203, 66 and 3 samples respectively. To conclude: The smears prepared from homogenised rat liver suspension and fixed like bacterial smears offer a very convenient and reliable tissue substrate for ANA test.