Developing the sensitive point-of-care testing(POCT)of oncogenic nucleic acids from human papillomavirus(HPV)infection is essential in preventing cervical can-cer,especially in resource-limited settings.Rolling circle...Developing the sensitive point-of-care testing(POCT)of oncogenic nucleic acids from human papillomavirus(HPV)infection is essential in preventing cervical can-cer,especially in resource-limited settings.Rolling circle amplification(RCA)is attractive in achieving POCT via nucleic acid-based aggregation under isothermal conditions.However,the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity.Here,a minimum secondary structured RCA technique(MSS-RCA)is developed by designing a unique circu-lar template,demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions.The amplifi-cation efficiency of MSS-RCA could be kinetically manipulated by controlling the secondary structure of the circular template.Introducing the invertase probe to MSS-RCA,HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter(PGM)with a sensitivity of 5 fM(50 zmol in 10µL).This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00%and negative predictive values of 96.00%(77.77%to 99.40%,95%CI).MSS-RCA-PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.展开更多
基金National Natural Science Foundation of China,Grant/Award Numbers:22174121,22211530067,T22507101802022 Natural Science Foundation of Guangdong Provincial Basic and Applied Basic Research Fund(Guangdong Hybribio)+4 种基金Shenzhen Bay Open Laboratory Fund 2021National Key Research and Development Program of China,Grant/Award Number:2020YFA0509400CUHK(SZ)-Boyalife Joint Laboratory of Regenerative Medicine Engineering FundCUHK(SZ)University Development Fund,Grant/Award Number:UDF01002012Shenzhen Key Laboratory of Functional Aggregate Materials,Grant/Award Number:ZDSYS20211021111400001。
文摘Developing the sensitive point-of-care testing(POCT)of oncogenic nucleic acids from human papillomavirus(HPV)infection is essential in preventing cervical can-cer,especially in resource-limited settings.Rolling circle amplification(RCA)is attractive in achieving POCT via nucleic acid-based aggregation under isothermal conditions.However,the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity.Here,a minimum secondary structured RCA technique(MSS-RCA)is developed by designing a unique circu-lar template,demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions.The amplifi-cation efficiency of MSS-RCA could be kinetically manipulated by controlling the secondary structure of the circular template.Introducing the invertase probe to MSS-RCA,HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter(PGM)with a sensitivity of 5 fM(50 zmol in 10µL).This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00%and negative predictive values of 96.00%(77.77%to 99.40%,95%CI).MSS-RCA-PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.
