Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange–high-performance liquid chromatography (AE–HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2×10¹⁰ and 7×10¹¹ VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14×10⁹ VP/ml and a slope of 3.04×10⁵ VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.     

Uptake and translocation of zinc absorbed through roots and fruiting organs in peanuts

Peanut plants (Arachis hypogaea L.) are known to absorb Ca, P and S through the fruiting organs, but information on Zn uptake pattern is lacking. Therefore, a green-house experiment was conducted to study the uptake and translocation of Zn when applied in the rooting and fruiting zones of peanut plants. To locate the pathway and distribution of radioactive Zn, autoradiographs of plants were also taken.Zinc uptake data and autoradiographs indicated that a substantial amount of⁶⁵Zn was absorbed through the fruiting organs (auxillary system). Of the total⁶⁵Zn in the whole plant, 55.2 per cent was absorbed through roots and remaining 44.8 per cent through fruiting organs. Zinc was translocated to all the plant parts regardless of its absorption through roots or fruiting organs. The highest zinc concentration was recorded in the kernels, followed by leaves, stem and the shell.Contribution from the Department of Soils, Haryana Agric. Univ., Hissar-India.     

Limitations of conventional regression analysis a proposed modification

Summary The conventional genotype-environment interaction analysis cannot detect the theoretically ideal genotype which has been defined as the one with relatively low sensitivity in the poor environments and high sensitivity in the favourable environments. The computation of separate regression coefficients on the two regions of the response curve has been suggested to detect such genotypes. This procedure is simple and more convenient than the complicated curvilinear regression analysis.     

Repeatability of stability estimators for downy mildew incidence in pearl millet

Summary Repeatability of mean downy mildew (Sclerospora graminicola (Sacc.) Schroet.) incidence, regression coefficients and deviation mean squares were investigated for 25 pearl millet (Pennisetum typhoides (Burm.) Stapf. & Hubb.) genotypes in 20 environments by correlating arrays of these stability parameters over subsets of the 20 environments arranged according to the year-wise, random, stratified and extreme methods of environmental division. Correlation coefficients between arrays of mean downy mildew incidence from different pairs of subsets ranged from 0.57 to 0.98 and those of deviation mean squares from 0.58 to 0.96 indicating good repeatability of these parameters. Arrays of regression coefficients from different subsets, on the other hand, showed correlation coefficients that ranged from –0.58 to 0.96. Apparently, the regression index of stability was not repeatable for the genotypes and environments studied. Therefore, in order to identify a widely adapted genotype, testing is required to be carried out over a wider range of environments.     

Degradation of Alachlor [2-chloro-N-(methoxymethyl)-2′,6′-diethylacetanilide] by soil fungi

Summary A Penicillium sp. and a Trichoderma sp. were isolated from a soil previously treated with alachlor which is commonly used as herbicide. These fungi were found to degrade alachlor and only one degradation product was observed after 15 days of incubation. Whereas two products were noticed after 30 days with both the test organisms. re]19740913     

Evaluation of commercial soil health tests using a medium-term cover crop experiment in a humid,temperate climate

Plant and Soil - Microbial-driven biogeochemical cycles of phosphorus (P) in wetlands subjected to global climate warming result in a downstream eutrophication risk. However, how warming influences...     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.