The rapid detection of human HLA-B*27 gene based on BASIC isothermal detection

Figures

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  Fig. 1. BASIC scheme and primer design. (A) Schematic illustration of the BASIC assay. (B) The OTA of each primer. (C) The delta OTA of each primer. Data was from Fig. 1B. (D) BASIS amplification with Brip addition. (E) PAGE analysis of primer 7 amplicons. Original gel image is presented in Information. *P < 0.05, by Student’s t-test.

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  Fig. 2. Pathogenic HLA-B*27 detection. (A) HLA-B*27 genotypes alignment and gRNA design. HLA-B*27:05 in this work represents the HLA-B*27:05:02 allele. The red base represents the additional mismatch base in gRNA, which could improve the recognition ability of Cas12a. (B) BASIS amplification for pathogenic and non-pathogenic HLA-B*27 genes. (C) Real-time fluorescence analysis of BASIC. (D) Endpoint fluorescence analysis for BASIC. (E) CRISPR/Cas12a cleavage for HLA-B*27:05 amplicons. The cleaved product was analyzed by PAGE. Original gel image is presented in Supplementary Information.

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  Fig. 3. Optimization of the BASIC system. (A) BASIS amplification with 1 µM, 0.5 µM, 0.25 µM or 0.1 µM of primers. (B) BASIS amplification at 63, 64, 65 and 66 °C. (C) The CRISPR/Cas12a reaction time optimization. (D) The CRISPR/Cas12a concentration optimization.

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  Fig. 4. Performance of BASIC assay. (A) Sensitivity of BASIS. The sample concentrations were as follows: 107, 106, 105, 104, 103, 102, 101 copies/µL and negative control (without the template). (B) BASIC sensitivity for amplicons from Fig. 4A. (C) Specificity analysis of BASIC with a series of homologous genes. (D) The interference resistance performance of the BASIC. (E) Repeatability of BASIS. (F) BASIC repeatability based on Fig. 4E.

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  Fig. 5. Clinical validation of the BASIC. (A) Clinical samples detection by BASIC. The cut-off is defined as the standard above-mentioned in Fig. 2D. (B) Clinical samples detection by q-PCR. The positive is defined as Ct < 40 and the Ct of negative is defined as 40. Ct: The cycle threshold.