基于量子点微球和RPA技术的ASFV抗体-核酸快速现场联检方法的建立

doi:10.3864/j.issn.0578-1752.2024.24.013

Abstract

Abstract:

【Background】African swine fever (ASF), recognized as a notifiable disease by the World Organization for Animal Health (WOAH) and categorized as a Class I animal disease in China, presents significant challenges due to the absence of effective vaccines and treatment options. Therefore, early, effective, rapid, and sensitive detection on ASF is crucial for controlling ASF. Current clinical diagnostic methods for African swine fever virus (ASFV) primarily include quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). However, these techniques require complex procedures, expensive equipment, and extended detection times, rendering them unsuitable for rapid field testing. Moreover, the levels of antibodies and nucleic acids in the host exhibit significant variations over time following ASFV infection, which can result in false negatives if only ASFV nucleic acids or antibodies are tested in isolation. 【Objective】This study aimed to develop a test strip that allowed for the simultaneous detection of ASFV antibodies and nucleic acids based on quantum dot microspheres and recombinase polymerase amplification (RPA) technology. The objective was to enable on-site detection of both ASFV antibodies and nucleic acids, thereby enhancing the sensitivity and specificity of the tests, reducing detection costs, and saving time. 【Method】In this study, a lateral flow immunoassay strip for the simultaneous detection of ASFV antibodies and nucleic acids was developed by combining quantum dot fluorescence immunochromatography technology with recombinase polymerase amplification (RPA). The corresponding portable fluorescence analyzer enables rapid on-site dual detection of ASFV antibodies and nucleic acids. The study optimized key parameters, such as the quantum dot-protein conjugation amount and the coating concentration of the test line. Based on these optimizations, the cutoff value, sensitivity, specificity, and reproducibility of the detection method were determined. Furthermore, the developed ASFV dual detection strip was compared with commercially available ELISA and qPCR kits by testing clinical samples to assess detection consistency, validating the method's concordance, and evaluating the practical applicability of the test strip. 【Result】The dual detection strip was user-friendly and rapid, with the entire testing process completed within 30 minutes. It demonstrated no cross-reactivity with six other common porcine infectious viruses, confirming excellent specificity. The sensitivity for nucleic acid detection reached 10 copies/µL, while the sensitivity for antibody detection was up to 1﹕3 200. The intra-assay variability coefficients for both antibody and nucleic acid tests were less than 10%, and the inter-assay variability coefficients were less than 15%, indicating excellent reproducibility. Validation against commercial ELISA and qPCR kits yielded a conformity rate of 100%. Furthermore, in clinical sample testing, two samples tested positive for antibodies but negative for nucleic acids in the corresponding pigs, which exhibited no significant clinical symptoms. These findings further confirmed that the combined detection of antibodies and nucleic acids could enhance the accuracy of testing and reduce the incidence of false negatives. 【Conclusion】The cost per experiment for this nucleic acid-antibody dual detection method was lower compared with the total cost of separate nucleic acid and antibody testing, thus reducing both detection costs and time. It was sensitive to both acute outbreaks and chronic infections of ASF, addressing the limitations of detecting only nucleic acids or antibodies individually. The method had minimal requirements for operating conditions, and the portable fluorescence analyzer was easy to use, requiring neither expensive laboratory equipment nor highly specialized personnel. This reduced the precision demands on detection devices, making it suitable for ASFV testing in remote areas and at the grassroots level. Therefore, this method held great potential as a reliable tool for point-of-care ASFV detection in pig farms in the future.

Key words: African swine fever, nucleic acid detection, antibody detection, lateral flow immunochromatography test strip

ZHAO YiRan, SHAN YanKe, LI JiaHao, HE ZhaoQun, WANG XinYi, WEN Dun, WANG MiLa, CHU Rui, ZHAO DongMing, LIU Fei. Establishment of Rapid Field Co-Detection Method of ASFV Antibody and Nucleic Acid Based on Quantum Dot Microspheres and RPA Technology[J].Scientia Agricultura Sinica, 2024, 57(24): 4990-5002.

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引物/探针 Primer/Probe	序列Sequence（5′-3′）
引物F Primer F	TGATAGACCCCACGTAATCCGCGTAATCCGTGTCCCAAC
生物素-引物R Biotin-Primer R	biotin-GTTTCCATCAAAGTTCTGCAGCTCTTACATA
FITC-探针 FITC-Probe	FITC-CTCCCGTGGCTTCAAAGCAAAGGTAATCAT-THF-ATCGCACCCGGATCATCG-C3-spacer

样品 Sample	批内 Intra-assay			批间 Inter-assay
样品 Sample	1:200	1:400	1:800	1:200	1:400	1:800
T/T+C	0.44772	0.39053	0.30973	0.396821	0.38822	0.33565
	0.41416	0.37078	0.33944	0.477115	0.32101	0.32191
	0.43146	0.37649	0.32818	0.452673	0.36206	0.31119
变异系数CV	3.89%	2.68%	4.60%	9.31%	9.49%	3.80%

样品 Sample	批内 Intra-assay			批间 Inter-assay
样品 Sample	10⁷	10⁵	10³	10⁷	10⁵	10³
T/T+C	0.96707	0.71001	0.35254	0.94447	0.71428	0.31767
	0.98511	0.69655	0.29571	0.86099	0.64785	0.38631
	0.96146	0.68993	0.35298	0.96672	0.63711	0.29283
变异系数CV	1.27%	1.46%	9.87%	6.03%	6.27%	14.57%

Establishment of Rapid Field Co-Detection Method of ASFV Antibody and Nucleic Acid Based on Quantum Dot Microspheres and RPA Technology

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检测方法 Detection method	阳性样品数 Number of positive samples	阴性样品数 Number of negative samples	符合率 Concordance rate
试纸条 Test strip	21	52	100%
酶联免疫吸附试验 ELISA	21	52	100%

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