Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

Aim: Several methods described previously for isolation and purification of soil DNA. Most of these protocols use combination of techniques or methods but the role and contribution of each individual method or component used is not clearly discussed. This study aims at analysing the effect of individual components used in extraction of DNA from soil and finally to optimize soil DNA isolation protocol and its validation by using 16SrDNA sequence analysis. Methods and Results: The soil was washed with anionic buffers before lysis step to reduce humic substances and release microbial cells from soil matrix, then the cells were lysed using combination of SDS, heating and vortexing and finally humic substances were removed using chemical flocculation. Pre-lysis washing of soil with 100 mmol l-1 Na2EDTA proved good for releasing microbial cells from soil matrix. Heating the soil sample at 75°C yielded good quantity (15.73 µg g-1 soil) DNA followed by 2% SDS (10.28 µg g-1 soil) and vortexing at 1400 rpm (8.94 µg g-1 soil). Combination of heating, SDS and vortexing yielded 25 µg DNA per gram of soil. Different concentrations of chemical flocculants like AlNH4(SO4)2, FeCl3, CaCl2 and MgCl2 were used to reduce humic substances. Flocculation with 100 mmol l-1 CaCl2 removed 5.2 mg humic substances without significant loss of DNA. 16S rDNA sequence analysis of DNA extracted from soil reveals presence of all the common soil bacterial species indicating the protocol is unbiased. Conclusion: Combination of chemical (SDS) and physical (heating and vortexing) methods yield good DNA whereas addition of enzyme (lysozyme) did not show significant effect on cell lysis. The digestion of isolated DNA with restriction enzyme and amplification of 16S rDNA using Taq DNA polymerase indicates the isolated DNA is pure enough for metagnomic analysis. 16Sr DNA sequencing of soil DNA indicates that this protocol can extract good quality and quantity DNA from range of bacteria present in soil varying in their cell wall composition. The optimised protocol is unbiased, very simple, does not need special equipments and many samples can be processed simultaneously.

Assessment of cultural beliefs and practices during the postnatal period in an urban field practice area of SRMC, Nandyal, Kurnool, Andhra Pradesh

Background: The postpartum period continues to be an important part of the tradition and culture among Indian women. But frequently the health of the postnatal women is neglected. So, the present study aimed to explore the beliefs and practices in the postpartum period regarding diet, rest, hygiene, confinement and assess association between cultural practices and socio demographic characteristics.Methods: A cross‐sectional descriptive study was conducted in the field practice area of urban SRMC Nandyal. The participants were women who had given births in the past three months. The data was collected using a pre‐tested semi‐structured questionnaire.Results: Among the 140 women, over 75% of women had increased their diet intake postpartum. Vegetables such as brinjal and fruits like papaya were avoided by 58.5% and 63.6% women respectively. Among the mothers 18.3% consumed less than 500 ml of water every day and 22% did not drink milk at all. Household work was avoided by 67% of the women while 79.6% avoided going outdoors. Many women didn’t maintain personal hygiene. Many women took home remedies for faster recuperation. These practices were influenced by the socioeconomic status and the woman’s educational status.Conclusions: Traditional postpartum practices are still popular among women in rural and slum areas in Andhra Pradesh. It is critical to identify the harmful practices and reinforce the positive healthy practices to make postpartum period a healthy and joyful period for the mother.

Prevalence of Helicobacter pylori infection among low socio-economic workers.

Epidemiological studies have shown that the prevalence of Helicobacter pylori infection in a community and occupational health are closely related to lifestyle and socio-economic status. There is little information on H. pylori profile in industrial workers in the literature. The aim of this study was to investigate the prevalence rate of H. pylori profiles among low socio-economic workers in the United Arab Emirates (UAE). This study was undertaken by determining IgG H. pylori antibody profiles among industrial exposed and referent workers, sera. Presence of anti-H. pylori antibodies in the frozen stored sera was determined by ELISA. Also, data on dietary and lifestyle were obtained. The result was considered positive if IgG anti-H. pylori antibody titers was > 300. People with seropositive levels of IgG antibodies to H. pylori were assumed to be infected with H. pylori. Most of the industrial workers lived in less modern accommodation, were less educated, ate their vegetable products unwashed and did not have drinking water facilities, when compared to referents. H. pylori serology by IgG was positive in 167 industrial workers (78.4%) and 137 in referent workers (64.3%) respectively, (p < 0.002). The sensitivity and specificity of the IgG serology assay were 94.5%, and 97.2% respectively. There was statistically significant difference between the exposed industrial and non-exposed control groups in respect of their H. pylori profiles.

Variations in angiotensin-converting enzyme gene insertion/deletion polymorphism in Indian populations of different ethnic origins.

The pattern of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the Indian population is poorly known. In order to determine the status of the polymorphism, young unrelated male army recruits were screened. The population had cultural and linguistic differences and lived in an environment that varied significantly from one region to another. Analysis of the genotype, showed higher frequency of the insertion allele in four of the five groups i.e. I allele frequency was significantly higher (P < 0.05) in Dogras, Assamese and Kumaonese. The deletion allele frequency was comparatively higher in the fifth group that belonged to Punjab. A correlation was observed between the genotype and enzyme activity. Involvement of a single D allele in the genotype enhanced the activity up to 37.56 3.13%. The results suggested ethnic heterogeneity with a significant gene cline with higher insertion allele frequency. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics.