Rapid detection of the GJB2 c.235delC mutation based on CRISPR-Cas13a combined with lateral flow dipstick.

Abstract

Hereditary hearing loss, an auditory neuropathy disorder, is characterized by its high prevalence and significant impact on the quality of life of those affected. In Chinese populations, the most prevalent gap junction beta-2 (GJB2) mutation hotspot is c.235delC. Currently available genetic tests require expensive instruments and specialized technicians or have long testing cycles and high costs, and therefore cannot meet point-of-care testing (POCT) requirements. The objective of this study was to evaluate the viability of a POCT kit. In only 42 min, we successfully identified the GJB2 mutation site c.235delC by integrating CRISPR-Cas nucleic acid detection with recombinase-aided amplification (RAA) and a lateral flow dipstick (LFD) method. This method has the capacity to detect low-abundance nucleic acids (as low as 10² copies/μL) and low mutation frequency (20%), in addition to accurately distinguishing wild-type, homozygous, and heterozygous mutation. This approach was utilized to assess blood samples from a total of 31 deaf patients and 5 healthy volunteers. All results were subsequently confirmed through the implementation of Sanger sequencing. Our detection results were consistent with Sanger sequencing results. The diagnostic sensitivity and specificity were 100%. The combination of CRISPR-Cas13a and LFD may be a promising method for POCT of deafness genes.