Evaluation of DNA extracted from blood filter spots and eluates processed for enzyme linked immunosorbent assay (ELISA)

Abstract

15Dried filter blood spots have become a significant blood collection method for screening 16 individuals for clinical purposes. When used for ELISAs, they are normally discarded after the 17 blood has been eluted. However, they may still be useful for extraction of DNA for molecular-18 based assays. The aim of this work was to determine the integrity of DNA extracted from filter 19 paper spots from which blood has initially been eluted for ELISA with sample dilution buffer 20 (SDB) and phosphate buffered saline (PBS). DNA was extracted from the eluted filter spots, the 21 eluate, and dried blood filter spots (controls) using spin column extraction. The quality and 22 quantity of the extracted DNA was assessed and used for PCR to further evaluate their 2 23 usefulness in molecular assays. Concentration of DNA obtained was dependent on the buffer 24 used for processing the filter blood blots. Accounting for the DNA concentration obtained from 25 dried blood spots, which were used as controls, DNA extracted from the already eluted blood 26 spots were 32 times higher in PBS than SDB processed filter paper. The ratio was even higher 27 for the eluates, which were 57 times higher in PBS than SDS eluates. SDB eluates had 28 significantly higher average DNA concentration than their eluted filter paper, but their purity 29 ratios were similar. 85% PCR success rate was achieved with the DNA samples. Useful DNA 30 can be extracted from blood spots after it has been eluted with SDB. Although the DNA 31 concentration and purity may be low, the DNA could be useful for rather simple PCR assays. 32 Author Summary 33Collection of blood onto filter paper has become an accepted method for screening individuals 34 for clinical and public health purposes since the 1960s. This method of blood collection has 35 become increasingly popular due to its ease and convenience in collection and transportation. 36The use of dried blood spots for clinical evaluations and research has become very significant. 37For research purposes, DBS when used for ELISAs are discarded after single use. DNA may 38 however be extracted from the used filter blots and used for molecular assays. The concentration 39 of DNA obtained may be low but simple assays like PCR could be done using the DNA 40 extracted from the eluted filter spot. 41 96 paper). DNA was extracted from the eluate, eluted filter paper and the dried unprocessed DBS 97 using the Qiagen DNA Blood and Tissue kit according to the manufacturer's instruction. DNA 98 was eluted in 150 µL of buffer AE. DNA quantity and purity were measured using Qubit ® 2.0 6 99 fluorometer (Invitrogen, Life technologies) and NanoDrop 2000c Spectrophotometer (Labtech 100 International Ltd, Thermo Scientific, UK). Samples were stored at −40°C for further analysis. 101 Amplification of human ribosomal gene 102 PCR was performed to evaluate the quality of genomic DNA extracted. A 231bp region of the 103 small subunit of human ribosomal gene (ssrDNA) was amplified [16,17] UNR-HUF, 5'-104 GAGCCGCCTGGATACCGC-3' REV, ...