RPA-CRISPR/Cas12a结合重力驱动微流控芯片的MTB快检方法的建立

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

生物技术通报 ›› 2025, Vol. 41 ›› Issue (5): 62-69.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0955

RPA-CRISPR/Cas12a结合重力驱动微流控芯片的MTB快检方法的建立

高畅¹(), 庄添驰², 李宁³, 刘云⁴, 顾鹏飞¹, 赵昕怡⁵, 季明辉²()

^1.南京医科大学第二临床医学院，南京 211166
^2.南京医科大学护理学院，南京 211166
^3.南京市江宁区疾病预防控制中心，南京 211100
^4.南京市秦淮区疾病预防控制中心，南京 210001
^5.南京医科大学第一临床医学院，南京 211166

收稿日期:2024-09-30 出版日期:2025-05-26 发布日期:2025-06-05
通讯作者: 季明辉，男，博士，副教授，研究方向：病原体检测；E-mail: jiminghui@njmu.edu.cn
作者简介:高畅，女，研究方向：病原体检测；E-mail: 1975480691@qq.com
基金资助:
?：江苏省卫生健康委员会预防医学科研课题面上项目(Ym2023064);国家级大学生创新创业训练计划基金项目(202410312005Z);南京预防医学科研课题(NJYFKT202409);江苏高校优势学科建设工程四期项目“护理学”(苏教研函〔2023〕11号)

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

GAO Chang¹(), ZHUANG Tian-chi², LI Ning³, LIU Yun⁴, GU Peng-fei¹, ZHAO Xin-yi⁵, JI Ming-hui²()

^1.Second Clinical Medical College, Nanjing Medical University, Nanjing 211166
^2.School of Nursing, Nanjing Medical University, Nanjing 211166
^3.Jiangning District Center for Disease Control and Prevention, Nanjing 211100
^4.Qinhuai District Center for Disease Control and Prevention, Nanjing 210001
^5.First Clinical Medical College, Nanjing Medical University, Nanjing 211166

Received:2024-09-30 Published:2025-05-26 Online:2025-06-05

摘要/Abstract

摘要：

目的开发一种快速高效的结核分枝杆菌（MTB）检测方法，给基层单位、偏远地区的结核病（Tuberculosis）快速筛查提供技术方案，更好地开展结核病的防治工作。方法设计RPA引物、crRNA，并引入重力驱动的微流控芯片，建立RPA-CRISPR/Cas12a的片上检测方法，进一步分析该方法的灵敏度与特异性，使用疑似结核病患者的样本进行片上检测和痰培养，比较建立的方法与痰培养法的一致性。结果建立的RPA-CRISPR/Cas12a片上检测方法可在30 min内实现MTB检测，LOD为1 copies/μL。以痰培养鉴定为参考标准，片上检测法的灵敏度为91.11%，特异度为94.34%，阳性预测值为93.19%，阴性预测值为92.59%，准确性为92.86%，Kappa值为0.856。结论建立的RPA-CRISPR/Cas12a的片上检测法的灵敏度、特异性高，并且快速、简单、便捷。

关键词: 结核分枝杆菌, 重组酶聚合酶扩增, CRISPR/Cas12a, 微流控芯片, 快速检测

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

高畅, 庄添驰, 李宁, 刘云, 顾鹏飞, 赵昕怡, 季明辉. RPA-CRISPR/Cas12a结合重力驱动微流控芯片的MTB快检方法的建立[J]. 生物技术通报, 2025, 41(5): 62-69.

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.

图/表 5

表 1 RPA 引物、crRNA和探针序列

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

图1 针对结核分枝杆菌IS6110基因的RPA引物、crRNA筛选A：琼脂糖凝胶电泳检测来自不同引物的RPA扩增产物；B：通过琼脂糖凝胶电泳检测RPA扩增的特异性（1：结核分枝杆菌；2：金黄色葡萄球菌；3：铜绿假单胞菌；4：肺炎克雷伯菌）；C：不同crRNA的CRISPR/Cas12a反应的荧光强度；每次测试重复进行3次，荧光强度在图中显示为平均值±SD （NTC：阴性对照，ddH2O；* P<0.05，** P<0.01，**** P<0.000 1，下同）

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)

图2 RPA-CRISPR/Cas12a体系测试A：结核分枝杆菌的IS6110基因的RPA-CRISPR/Cas12a的特异性评估；B：结核分枝杆菌的IS6110基因的RPA-CRISPR/Cas12a的灵敏度评估；每次测试重复进行3次，荧光强度在图中显示为平均值±SD

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

图3 RPA-CRISPR/Cas12a检测的重力驱动微流控芯片A：芯片结构示意图；B：芯片使用原理图；C：芯片实物图；D：使用染料进行芯片测试图；E：使用芯片进行RPA-CRISPR/Cas12a片上检测结果图

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

表2 RPA-CRISPR/Cas12a片上检测和痰培养方法对痰液样本中的结核分枝杆菌检测结果

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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RPA-CRISPR/Cas12a结合重力驱动微流控芯片的MTB快检方法的建立

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

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