ABSTRACT
Amplification of influenza A virus gene segments by reverse transcription-polymerase chain reaction (RT-PCR) can be combined with enzymatic digestion to reveal unique restriction fragment length polymorphisms specific for H1N1 and H3N2 subtype viruses. We have used the method to provide a rapid, specific and reproducible identification of the genotype of high-growth influenza reassortants derived from A/Puerto Rico/8/34 (PR8). Digestion of the gene segments amplified from wild-type viruses, PR8 and reassortants at sites unique to either the wild-type strain or to PR8 provided positive, unambiguous identification of the origin of each of the internal genes, and distinguished the internal genes of both H1N1 and H3N2 strains from those of PR8. This method has also permitted us to quickly confirm that reassorting has occurred and to optimize the selection of reassortant clones with maximum number of PR8 internal genes. Since the method can detect 1-10% of a second strain in a mixed population, the method can also be used to detect samples containing more than one viral subtype and to assess the purity of influenza viruses used for manufacturing vaccines.
Subject(s)
Genes, Viral , Influenza A virus/classification , Influenza A virus/genetics , Polymorphism, Restriction Fragment Length , Reverse Transcriptase Polymerase Chain Reaction , DNA, Complementary , DNA, Viral/genetics , DNA, Viral/isolation & purification , DNA, Viral/metabolism , Genotype , Humans , Influenza Vaccines , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reassortant Viruses/classification , Reassortant Viruses/genetics , Recombination, Genetic , Reproducibility of Results , Restriction Mapping , Sensitivity and SpecificityABSTRACT
Sixty nine samples of maize were collected from pre-harvest standing crops and on-farm storage facilities from 52 smallholder farms located within 4 regions of Honduras during October 1992 and November 1993. Samples were visually assessed for insect damage and fungal spoilage, and the mycoflora quantified on artificial media. The major components of the ear rot complex were: Fusarium moniliforme, F. moniliforme var. subglutinans, Penicillium species, Stenocarpella maydis, S. macrospora and Acremonium spp. Representative samples were also assayed for mycotoxin content. Fumonisin B1 was detected in all 24 samples tested at levels of between 68-6,555 (micrograms/kg), and aflatoxin was detected in 2 samples heavily contaminated with Aspergillus flavus. Moniliformin and tenuazonic acid were not detected in the samples tested. The implications of these findings for human and livestock health risk are discussed, together with possible strategies for controlling these pathogens.
Subject(s)
Food Microbiology , Fumonisins , Mitosporic Fungi/isolation & purification , Mycotoxins/analysis , Zea mays/microbiology , Aflatoxins/analysis , Cyclobutanes/analysis , Honduras , Tenuazonic Acid , Zea mays/chemistryABSTRACT
High-performance thin-layer chromatography (HPTLC) was applied to the separation and quantification of aflatoxin in 300 jars of "crunchy" peanut butter. A critical evaluation of the proposed HPTLC method has been carried out by statistical comparisons with a commercially available enzyme-linked immunosorbent assay (ELISA) kit and a high-performance liquid chromatographic (HPLC) method. The statistical tests indicated that whilst the distributions of the data sets obtained with each method were similar, the HPLC method was found to be biased. Over-all results indicated that the HPTLC method gave more consistent data, relatively lower standard deviations and lower coefficients of variation. The ELISA kit was found to be less precise than the HPTLC and HPLC methods and prone to some loss of sensitivity caused by matrix interference.
