Visual detection of Coxsackievirus A6 using a reverse transcription polymerase spiral reaction method

Kaifeng Wu^1*Kun Wang¹Yuanhang Ai¹Juan Luo¹Longying Liang¹Weiwei Zhang¹Guojun Cao²He Zha¹Jie Wu¹Kun Lei¹Shifei Yao^1*

• ¹Zunyi Medical University Third Affiliated Hospital, Zunyi, China
• ²Huashan Hospital, Fudan University, Shanghai, Shanghai Municipality, China

Coxsackievirus A6 (CVA6) ranks as a primary enterovirus associated with handfoot-mouth disease (HFMD) and herpangina (HA). Given its significant role in these diseases, there is an urgent need for an efficient identification method. This study presents a novel visual approach based on the reverse transcription polymerase spiral reaction (RT-PSR) for the rapid detection of CVA6. We designed an RT-PSR assay that targets and amplifies a segment of the VP1 gene. Hydroxy naphthol blue (HNB) is incorporated as the detection agent in this assay. To evaluate the performance of the RT-PSR assay, we analyzed 142 clinical throat swab samples. The results were benchmarked against those obtained using quantitative reverse transcriptionpolymerase chain reaction (qRT -PCR). The RT-PSR assay operates at 65º C for 60 minutes and exhibits a detection limit of 10 copies/μL. When tested against other viruses, it consistently yielded negative results, demonstrating its high specificity.Moreover, the RT -PSR assay showed excellent agreement with a commercial qRT -PCR kit. In conclusion, by using HNB as an indicator, the RT -PSR assay emerges as a straightforward and highly sensitive method for detecting CVA6 in symptomatic throat samples. This approach holds great potential for improving the diagnosis and surveillance of CVA6 -related diseases.