Additional file 1 of A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay
Creators: name: Liu, Yan
name: Chao, Zhujun
name: Ding, Wei
name: Fang, Tanfeng
name: Gu, Xinxian
name: Xue, Man
name: Wang, Wei
name: Han, Rong
name: Sun, Wanping
Publisher: name: figshare
Year: 2024
Subjects: subject: Space Science
subject: Biotechnology
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Cancer
subject: Science Policy
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Table S1. Sequences of plasmids. Table S2. Sequences of primers. Table S3. Coverage of HPV 16/18/31/33/35/45 crRNAs used for the H-MRC12a. Figure S1. H-MRC12a method detection results and sequencing alignment of the RPA amplification product for an HPV 31-positive clinical sample not detected by the QPCR method. a The sample, after heat inactivation, underwent nucleic acid extraction using the nanomagnetic bead method. The extracted nucleic acids were incubated in the H-MRC12a combined detection system for 40 mins (Multiple RPA incubation for 20 mins, followed by CRISPR incubation for an additional 20 mins) and immediately observed under 300 nm UV light. b Real-time fluorescence signals (recorded using the Q160 LongGene portable QPCR instrument) of the clinical sample and negative control after 20 mins of incubation in the CRISPR step. c Bar chart of the fluorescence endpoint values for the clinical sample and negative control after 20 mins of incubation in the CRISPR step. d Sequencing results of the RPA amplification product for the clinical sample. e Alignment of the base sequence obtained from sequencing of the clinical sample with the NCBI database. The comprehensive results indicate a high similarity between the RPA amplification product sequence of the sample and various HPV 31 sequences in the NCBI database. Figure S2. H-MRC12a method detection results and sequencing alignment of the RPA amplification product for an HPV 35-positive clinical sample not detected by the QPCR method. a The sample, after heat inactivation, underwent nucleic acid extraction using the nanomagnetic bead method. The extracted nucleic acids were incubated in the H-MRC12a combined detection system for 40 mins (Multiple RPA incubation for 20 mins, followed by CRISPR incubation for an additional 20 mins) and immediately observed under 300 nm UV light. b Real-time fluorescence signals (recorded using the Q160 LongGene portable QPCR instrument) of the clinical sample and negative control after 20 mins of incubation in the CRISPR step. c Bar chart of the fluorescence endpoint values for the clinical sample and negative control after 20 mins of incubation in the CRISPR step. d Sequencing results of the RPA amplification product for the clinical sample. e Alignment of the base sequence obtained from sequencing of the clinical sample with the NCBI database. The comprehensive results indicate a high similarity between the RPA amplification product sequence of the sample and various HPV 35 sequences in the NCBI database. Figure S3. QPCR amplification curves for eight clinical samples positive for HPV 16, labeled as S16-1 to S16-8. Figure S4. QPCR amplification curves for eight clinical samples positive for HPV 18, labeled as S18-1 to S18-8. Figure S5. QPCR amplification curves for three clinical samples positive for HPV 31, labeled as S31-1 to S31-3, and for HPV 33, 35, and 31/45 double-positive clinical samples labeled as S33-1, S35-1, and S31/45, respectively. Figure S6. QPCR amplification curves for clinical samples positive for various additional high/low-risk HPV types, detected by the H-MRC12a method. S39/53: HPV 31/53 double-positive sample. S51/52: HPV 51/52 double-positive sample. S58/6: HPV 58/6 double-positive sample. S68, S81, S82: Samples positive for HPV 68, 81, and 82, respectively.