A new simple, cost-effective, and noninvasive diagnostic test for sickle cell disease (SCD) has been developed, according to an article published in Diagnostics.
The new test utilizes buccal swabs with isothermal DNA amplification coupled with restrictase-mediated cleavage (iDAR) to identify SCD patients (SS alleles), SCD carriers (SA alleles), and healthy individuals (AA alleles).
“Existing genetic diagnostic tests based on [polymerase chain reaction (PCR)], such as DNA sequencing and [quantitative PCR], typically require a complicated operational procedure, as well as expensive and sophisticated instruments that may not be available in many laboratories, whereas the iDAR assay can be done with less complex instruments and provides reliable, inexpensive, easy-to-use, and affordable genetic diagnostics for SCD, facilitating the expansion of SCD screening in low-resource settings,” the authors explained.
The iDAR assay was found to have a sensitivity of 91.5%, specificity of 100%, and 100% concordance with Sanger sequencing. The process was also found to have a limit of detection between 10 and 100 target DNA copies.
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The assay is cost-effective and time-saving, only costing about $5 and taking an estimated 150 minutes. Once the buccal swab has been collected, the sample is subjected to DNA extraction with the use of a lysate buffer for 20 minutes. Next, isothermal DNA amplification is performed for 20 minutes to amplify the DNA of interest. Targeted cleavage utilizing the Eco81I restriction enzyme occurs for 1 hour before the samples are finally run on gel electrophoresis.
The Eco81I enzyme targets the CCTNAGG sequence (where N could be any nucleotide) on the HBB gene. SCD mutations alter the target sequence and eliminate the restriction enzyme site, leading to no cleavage of the samples.
On electrophoresis, the presence of 2 bands would show evidence of cleavage and indicate healthy individuals (AA). A single band would indicate SCD (SS) since no cleavage would have occurred, while the presence of 3 bands would indicate that only some DNA was cleaved for heterozygous individuals (SA).
The assay could also be carried out in a one-pot reaction, where only a single reaction tube could be utilized to avoid contamination and ease workflow.
Thakur P, Gupta P, Bhargava N, et al. A simple, cost-effective, and extraction-free molecular diagnostic test for sickle cell disease using a noninvasive buccal swab specimen for a limited-resource setting. Diagnostics (Basel). 2022;12(7):1765. doi:10.3390/diagnostics12071765