Gravity-driven Microfluidic Chip Based on RPA-CRISPR/Cas12a for the Rapid Detection of Mycobacterium tuberculosis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0955

Abstract

Abstract:

Objective To develop a rapid and efficient method for detecting Mycobacterium tuberculosis (MTB), providing a technical solution for rapid screening of tuberculosis (TB) in grassroots units and remote areas, thereby enhancing TB prevention and control efforts. Method RPA (recombinase polymerase amplification) primers and crRNA were designed, and a gravity-driven microfluidic chip was introduced to establish an RPA-CRISPR/Cas12a on-chip detection method. The sensitivity and specificity of this method were further analyzed using samples from suspected TB patients for on-chip detection and sputum culture, comparing the established method with the sputum culture method for consistency. Result The established RPA-CRISPR/Cas12a on-chip detection method achieves MTB detection within 30 min, with a limit of detection (LOD) of 1 copy/μL. Using sputum culture identification as a reference standard, the on-chip detection method demonstrated a sensitivity of 91.11%, specificity of 94.34%, positive predictive value of 93.19%, negative predictive value of 92.59%, and accuracy of 92.86%, with a Kappa value of 0.856. Conclusion The developed RPA-CRISPR/Cas12a on-chip detection method presents high sensitivity and specificity, characterized by rapid, simple, and convenient.

Key words: Mycobacterium tuberculosis, RPA, CRISPR/Cas12a, microfluidics chip, rapid detection

GAO Chang, ZHUANG Tian-chi, LI Ning, LIU Yun, GU Peng-fei, ZHAO Xin-yi, JI Ming-hui. Gravity-driven Microfluidic Chip Based on RPA-CRISPR/Cas12a for the Rapid Detection of Mycobacterium tuberculosis[J]. Biotechnology Bulletin, 2025, 41(5): 62-69.

Figures/Tables 5

Table 1 Primers, crRNA and ssDNA reporter used in this study

名称 Primer	序列 Primer sequence （5′-3′）	长度 Length （bp）
F1	GTCATGTCAGGTGGTTCATCGAGGAGGTACC	31
R1	GCCCACTCCGAATCGTGCTGACCGCGGATCT	31
F2	GTCATGTCAGGTGGTTCATCGAGGAGGTACC	31
R2	TGCCCACTCCGAATCGTGCTGACCGCGGATC	31
F3	GGTCATGTCAGGTGGTTCATCGAGGAGGTAC	31
R3	TGCCCACTCCGAATCGTGCTGACCGCGGATC	31
F4	GTCATGTCAGGTGGTTCATCGAGGAGGTACC	31
R4	CCCACTCCGAATCGTGCTGACCGCGGATCTC	31
crRNA-1	GATGAGCCCGCGGCCTATCAGCTTG	25
crRNA-2	TGGTGGGGTGACGGCCTACCAAGGC	25
crRNA-3	ACGACGGGTAGCCGGCCTGAGAGG	24
crRNA-4	GGAGGTGGAGAAGAAGCACCGGCC	24
ssDNA报告探针	6-FAM-TTATT-BHQ-1	5

Fig. 1 Screening of RPA primers and crRNA targeting the IS6110 gene of M. tuberculosisA: Detection of RPA amplification products from different primers by agarose gel electrophoresis. B: Detection of RPA amplification specificity by agarose gel electrophoresis (1: Mycobacterium tuberculosis; 2: Staphylococcus aureus; 3: Pseudomonas aeruginosa; 4: Klebsiella pneumoniae. C: Fluorescence intensity of CRISPR/Cas12a reactions with different crRNAs. Each test was repeated three times, and fluorescence intensity is shown in the figure as mean±SD (NTC: negative control, ddH2O; * P<0.05, ** P<0.01, **** P<0.000 1. The same below)

Fig. 2 RPA-CRISPR/Cas12a system testingA: Specificity assessment of RPA-CRISPR/Cas12a targeting the IS6110 gene of M. tuberculosis. B: Sensitivity assessment of RPA-CRISPR/Cas12a targeting the IS6110 gene of M. tuberculosis. Each test was repeated three times, and fluorescence intensity is shown in the figure as mean±SD

Fig. 3 Gravity-driven microfluidic chip for RPA-CRISPR/Cas12a detectionA: Schematic diagram of the chip structure. B: Schematic diagram of the chip working principle. C: Image of the chip. D: Chip testing image using dye. E: Detection results of RPA-CRISPR/Cas12a assay on the chip

Table 2 Detection of M. tuberculosis in sputum samples using RPA-CRISPR/Cas12a and sputum culture methods

RPA-CRISPR/Cas12a片上检测 RPA-CRISPR/Cas12a for on-chip detection	痰培养 Sputum culture			灵敏度 Sensitivity （%）	特异性 Specificity （%）	阳性预测值 Positive predictive value （%）	阴性预测值 Negative predictive value （%）	准确性 Accuracy （%）	Kappa值
	阳性 Positive	阴性 Negative	合计 Total	灵敏度 Sensitivity （%）	特异性 Specificity （%）	阳性预测值 Positive predictive value （%）	阴性预测值 Negative predictive value （%）	准确性 Accuracy （%）	Kappa值
阳性 Positive	123	9	132	91.11	94.34	93.19	92.59	92.86	0.856
阴性 Negative	12	150	162
合计 Total	135	159	294

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