DEVELOPMENT AND APPLICATION OF REAL-TIME AND LATERAL FLOW DIPSTICK RECOMBINASE POLYMERASE AMPLIFICATION ASSAYS FOR RAPID SPECIES IDENTIFICATION OF PELTEOBAGRUS FULVIDRACO
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摘要:
研究以黄颡鱼为目标对象, 基于重组酶聚合酶扩增(Recombinase polymerase amplification, RPA)等温扩增技术, 开发了实时荧光型RPA和侧向流RPA试纸条两种现场快速检测方法。这些方法能够用于快速、准确和灵敏地实时检测出黄颡鱼DNA。首先, 通过对黄颡鱼及其近缘物种的鱼类线粒体基因组序列进行对比分析, 针对COI基因保守区域, 设计了特异性探针和引物。测试结果显示, 在37℃条件下, 实时荧光型RPA和侧向流RPA试纸条都具有良好的特异性。实时荧光型RPA方法能够在20min内完成黄颡鱼DNA的检测, 且最低检测限可以达到102拷贝数/反应; 而侧向流RPA试纸条的操作更加便捷, 能够在10min内可完成对102低拷贝数的黄颡鱼DNA的扩增和鉴定。应用这两种RPA检测技术对149份长江流域常见鱼类进行现场检测, 两种方法均显示出较高的准确性(准确率100%)。因此, 研究开发的两种用于检测黄颡鱼的实时荧光型RPA和侧向流RPA试纸条方法, 拥有操作简便、结果直观、准确率高、不依赖实验室仪器设备等优势, 具有良好的现场快速检测应用前景。此外, 其克服了形态学物种鉴定专业门槛高、难以鉴定残破组织样品的问题, 检测靶标可跨越卵、鱼苗、成鱼等全生命周期, 为水产品监管执法提供技术手段。
Abstract:In order to effectively prevent illegal fishing of wild fishery resources in the Yangtze River, it is crucial to provide technical support for the “ten-year ban on fishing in the Yangtze River” and develop methods that can quickly and accurately identify fish species on site. This study focuses on Pelteobagrus fulvidraco and utilizes isothermal amplification technology, specifically Recombinase Polymerase Amplification (RPA), to develop two rapid on-site detection methods: Real-time fluorescent RPA and lateral flow RPA test strips, which are ideally suited for the swift in-field detection of P. fulvidraco DNA. We developed an RPA assay based on either Real-time fluorescent detection (Real-time RPA assay) or lateral flow dipstick (RPA-LFD assay) for rapid, accurate, and sensitive detection of P. fulvidraco DNA, facilitating effective management of the fishing ban in the Yangtze River basin. Primers and probes for both assays were designed to target conserved regions of the mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene in P. fulvidraco. Tests demonstrated that both the Real-time RPA and RPA-LFD assays exhibited good specificity at 37℃. The Real-time RPA assay achieved a detection limit of 102 copies of P. fulvidraco DNA per reaction, with amplification completed within 20min, though it requires a constant temperature amplification instrument. In contrast, the RPA-LFD assay offers a more convenient operation with a shorter amplification time of only 10min for 102 copies of P. fulvidraco DNA. Additionally, both methods were tested on 149 samples for known fish species, yielding results consistent with actual species identification. The Real-time fluorescence RPA and lateral flow dipstick RPA methods developed in this study for detecting P. fulvidraco DNA have the advantages of easy operation, intuitive results, high accuracy, and no dependence on laboratory instruments and equipment. They have good prospects for rapid on-site detection applications. In addition, it overcomes the problems of high professional threshold for morphological species identification and difficulty in identifying damaged tissue samples. The detection targets can span the entire life cycle of eggs, fish fry, and adult fish, providing essential technical support for aquatic product supervision and law enforcement.
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图 2 不同温度条件下的实时荧光型RPA反应
A. 37℃; B. 39℃; C. 41℃
Figure 2. Optimization of Real-time fluorescent RPA reaction under different temperatures
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图 3 侧向流RPA试纸条的反应条件优化
A. 侧向流RPA试纸条在不同的温度条件下的实验结果; B. 侧向流RPA试纸条在不同反应时间的实验结果
Figure 3. Optimization of RPA-LFD assay
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图 4 实时荧光型RPA和侧向流RPA试纸条灵敏度试验结果
A. 实时荧光型RPA检测黄颡鱼DNA灵敏度; B. 侧向流RPA试纸条检测黄颡鱼DNA灵敏度结果; NC代表阴性对照
Figure 4. Sensitivity results of Real-time fluorescent RPA and RPA-LFD assay
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图 6 侧向流RPA试纸条特异性试验结果
1. 黄颡鱼; 2. 长须黄颡鱼; 3. 瓦氏黄颡鱼; 4. 光泽黄颡鱼; 5. 鲢; 6. 鳙; 7. 青鱼; 8. 草鱼; 9. 刀鲚; 10. 短额鲚; 11. 鳜; 12. 大眼鳜; 13. 翘嘴鲌; 14. 达氏鲌; 15. 蒙古鲌; 16. 红鳍原鲌; 17. 阴性对照
Figure 6. Specificity results of RPA-LFD assay
表 1 RPA引物探针设计与序列
Table 1 Primers and probes sequences
名称Name 基因序列Sequence (5′—3′) P. fulvidraco-Fe TCGTCGTGGCATGCCAGCTAAGCCTAGGAAGTG P. fulvidraco-Re CGTATTTGCCATCATAGGAGCCTTCGTACACTG P. fulvidraco-Pe CCAAAGTGGATTTTTGTTCAGGTGTCATGTA/i6FAMdT/T/idSp//iBHQ1dT/ATAGCCCGTAAATAG/iSpC3/ P. fulvidraco-Rn 5′Biotin-CTTCCACTACGTCTTATCAATGGGGGCCGTAT P. fulvidraco-Pn 5′FAM-TGTATAGCCCGTAAATAGGGGGAATCAGTGT[THF]ACGAAGGCTCCTATG-3′C3 Spacer 
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表 2 实时荧光型RPA和侧向流RPA试纸条对鱼类样本的检测结果
Table 2 Detection results of fish samples by Real-time fluorescent RPA and RPA-LFD assay
鱼类样本Fish sample 样品数Sample quantity 荧光RPA检测结果Results of Real-time fluorescent RPA 侧向流RPA试纸条检测结果Results of RPA-LFD assay 黄颡鱼Pelteobagrus fulvidraco 14 ＋ ＋ 长须黄颡鱼Pelteobagrus eupogon 6 － － 瓦氏黄颡鱼Pelteobagrus vachellii 6 － － 光泽黄颡鱼Pelteobaggrus nitidus 6 － － 鲢Hypophthalmichthys molitrix 20 － － 鳙Hypophthalmichthys Nobilis 14 － － 青鱼Mylopharyngodon piceus 6 － － 草鱼Ctenopharyngodon idella 12 － － 刀鲚Coilia nasus 7 － － 短额鲚Coilia brachygnathus 6 － － 鳜Siniperca chuatsi 14 － － 大眼鳜Siniperca knerii 6 － － 翘嘴鲌Culter alburnus 14 － － 达氏鲌Chanodichthys dabryi 6 － － 蒙古鲌Chanodichthys mongolicus 6 － － 红鳍原鲌Culter erythropterus 6 － － 注: “＋”代表检测结果阳性, “－”代表检测结果阴性 Note: “＋” represent the positive results, “－” represent the negative results.
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