Development of a multiplex recombinase amplification assay for the rapid and concurrent detection of human DNA and...

In forensic practice, it is essential to identify human DNA and determine the sex of individuals from biological samples collected at crime scenes. Currently, the common detection methods mainly focus on targeted DNA analysis based on PCR technology, which is time-consuming and relies on laboratory equipment. In recent years, recombinase polymerase amplification (RPA), as one of ubiquitous isothermal amplification technology, has gained popularity across various diagnostic fields due to its advantages of rapid processing and minimal temperature control requirements. This study has developed a multiplex RPA assay suitable for human and sex components identification. The assay has good sensitivity (as low as 25 pg) and strong tolerance to inhibitors (in the presence of 200 ng/μL humic acid, 400 ng/μL tannic acid, and 8000 ng/μL collagen). Furthermore, we combined the alkaline lysis and RPA detection to construct a rapid detection scheme, which can shorten detection time to half an hour. We also conducted a preliminary exploration of the visualization scheme for the constructed RPA assay. The above research demonstrates simultaneous and rapid detection of human and sex components, offering an accurate and sensitive detection scheme.