SUCROSE DENSITY-GRADIENT ISOLATION METHOD INTENDED FOR IMPROVEMENT OF SAMPLE PURITY PRIOR TO AMPLIFICATION OF GIARDIA Β-GIARDIN GENE

Abstract

PCR-based methods have been widely used for detection of Giardia in stool. The sensitivity and hence degree of success of the molecular tool depends on many factors including efficiency of DNA extraction method, in addition to the degree of purity which necessitate removal of a great number of inhibitors from the stool samples. Therefore, Sucrose density gradient isolation method was assessed in this study to be applied prior to nucleic acid extraction and amplification of Giardia duodenalis in stool samples. The methods: Two different approaches were applied in this study to amplify P-giardin gene specific for Giardia duodenalis (syn. G. intestinalis, G. lamblia) using 30 microscopically Giardia positive fecal sam- ples. A) Direct nucleic acid extraction using QIAamp Mini spin columns B) Sucrose density-gradient isolation method prior to DNA extraction using the same extraction Kit. The property of the extracted samples was determined by Spectrophotometric analysis at 260 and at 280 nm. The results showed that in spite of the significant larger amount of nucleic acid obtained by method (A) versus method (B), more pure form of DNA was encountered by method (B) than method (A). Purity encountered by 'method (A) reflected protein contamination of the samples. Following nested PCR reaction, 28 samples of group (B) showed positive bands (511pb), while 8 samples from group (A) failed to be amplified.