环介导等温扩增技术快速检测水产动物病原的研究
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摘要
随着水产养殖规模的扩大和养殖环境的恶化,水产动物的病害越发严重,成为制约水产养殖业健康发展的瓶颈因素之一。水产病原的快速诊断,对水产养殖的良种选育,疾病预防、进出口检疫、水产品食品安全等方面具有重大意义。由于各自的缺陷,常规的病原检测技术大都不适合养殖基层的现场快速检测。环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)是一种新的DNA扩增方法。该技术依赖于自动循环的链置换反应,通过识别靶序列上6个特异区域的引物和具有链置换的DNA聚合酶,在等温条件下,不到1h就能扩增出109靶序列拷贝,具有特异、灵敏、快速、简便等特点,适合于养殖基层的现场快速检测。
本研究新建了环介导等温扩增方法分别快速检测对虾白斑综合征病毒、皮下及造血组织坏死病毒和弧菌。首次进行了白斑综合征病毒和皮下及造血组织坏死病毒进行同时检测的研究,以及首次对某一类特定病原体进行通用LAMP检测研究,研究了LAMP对弧菌属细菌的通用性检测。设计不同病原相应的检测引物,分别对其反应条件(镁离子浓度、甜菜碱浓度、反应时间、反应温度等)进行了优化,并检测了其特异性及灵敏度。首次将dUTP-UNG酶(尿嘧啶-N-糖基化酶)技术引入LAMP检测中,克服了LAMP反应极易产生假阳性的缺点,并对dUTP对dTTP的替代率、替代对反应灵敏度的影响进行了研究。并将建立好的LAMP检测方法用于实际水产动物样品的病原检测。具体结论如下：
1、优化后的白斑综合征病毒LAMP反应体系在64℃下45min即能有理想扩增效果。加入SYBR Green I后,能用肉眼进行结果的判断。使用常见细菌和对虾病毒做对照,显示LAMP反应对WSSV检测具有很好的特异性。灵敏度达到100copies/μl,比巢式PCR高100倍。反应中使用dUTP替代部分dTTP,结合UNG酶,可以避免LAMP的产物遗留污染的问题,但替代率高于50%后,显著抑制反应的扩增效率。在对虾样品的WSSV检测中,LAMP方法表现出比巢式PCR更好的检测灵敏度和更快速的检测流程。
2、皮下及造血组织坏死病毒LAMP反应体系在65℃反应60mmin完成扩增反应。产物经荧光染色后发生颜色变化,产物检测方便。对扩增产物进行酶切得到与预期相吻合的片段。50%的dUTP替代和JNG酶的使用能避免假阳性的发生,但会使反应灵敏度下降10倍。LAMP反应对IHHNV具有较好的特异性。其LAMP灵敏度较普通PCR高1000倍。对实际对虾样品的IHHNV检测中,LAMP方法于PCR方法结果一致,但表现出更高的效率和更加直观准确的结果。
3、优化后的WSSV和IHHNV多重LAMP反应体系需在65℃反应60min。产物经过酶切得到的与预期大小相吻合的条带。多重LAMP具有较好的特异性和较高的灵敏度,比巢式PCR高两个数量级,较普通PCR高三个数量级。在实际对虾样品的检测中,多重LAMP检测较PCR检测具有更高的病毒检出率和更高的检测效率。
4、优化后的弧菌属细菌通用LAMP检测的反应体系需在62℃反应60min。通用LAMP检测对弧菌属细菌具有高度的特异性。检测灵敏度较普通PCR方法高100倍。将通用LAMP检测方法应用于人工感染弧菌的水产动物检测上,表现了很好的检测效果。
检验结果表明,LAMP检测方法是一种操作简单、灵敏度和特异性较高的快速检测手段；无需特殊仪器设备,非常适合基层养殖场和防疫部门开展实地检测,同时也适合开发为快速检测试剂盒,在水产病原的实地快速高诊断方面具有重大的开发潜力。
With the scale expansion of aquaculture and degradation of aquatic environment, diseases of aquatic animals has become more and more serious, which is one of the bottlenecks of the aquaculture development. Rapid diagnosis of aquatic pathogens was of great significance for breeding, disease prevention, import and export quarantine, aquatic food safety, etc. Since their individual deficiency, most of the conventional pathogen detection techniques are not suitable for rapid field detection. Loop-mediated isothermal amplification (LAMP) is a new DNA amplification method. It relies on the automatic cycle of the strand displacement reaction. With a set of primers targeting six specific regions and DNA polymerase with strand displacement, it can amplify target nucleic acids from a few copies to109copies within1h. High specificity, sensitivity and efficiency, with easy protocol, make LAMP technique suitable for rapid field detection.
In this study, LAMP methods for rapid detection of white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Vibrios were developed. Multiplex LAMP for detecting WSSV and IHHNV at the same time and universal LAMP for detecting a group of specific pathogens were developed for the first time. The universal LAMP was used to detect Vibrio spp. Different primers were designed to targeting different pathogens DNA sequences. LAMP reaction conditions were optimized, including concentration of magnesium ions, the betaine concentration, reaction time, reaction temperature, etc. And the specificity and sensitivity were tested. DUTP-UNG (uracil-N-glycosylase) system was used to overcome carry-over contamination of LAMP for the first time. The replacement rate of dUTP to dTTP and the influence of the replacement to LAMP sensitivity were studied. LAMP detection systems were used for aquatic animal samples detection. Specific conclusions are as follows:
1. Incubating the optimized LAMP reaction for WSSV at64℃for45min can have the effect amplification. By adding SYBR Green I, the results can be judged by naked eyes. Using common bacteria and shrimp viruses as controls, LAMP method has good specificity WSSV detection. Sensitivity of LAMP was100copies/μl, which was100times higher than the nested PCR. DUTP replacing part of dTTP in the reaction mixture, combining the UNG enzyme, LAMP carry-over contamination can be prevented. But when the replacement rate was higher than50%, amplification efficiency was significantly inhibited. The LAMP method has higher sensitivity and faster detection process than nested PCR in shrimp samples detection against WSSV.
2. LAMP reaction system for IHHNV amplified properly at65℃for60min. Reaction tube color changed after staining, that makes it convenience for judgment. Expected fragments appeared after amplicons were digested by restriction enzyme. Fifty percent of the dUTP substitution and the using of the UNG can avoid the false positives, but that made the sensitivity decrease by about10times. LAMP detection showed good specificity for IHHNV. Its sensitivity was1000times higher than common PCR. In shrimp samples IHHNV detection, the LAMP method was consistent with PCR, but showed a higher efficiency and more intuitive results.
3. Optimized mLAMP system for WSSV and IHHNV detection was incubated at65℃for60min to complete the reaction. After digestion, fragments of amplicons were consistent with the expectation. Multiplex LAMP has high specificity and sensitivity, which was100times higher than nested PCR and1000times higher than the conventional PCR, respectively. Multiplex LAMP detection showed higher detection rates and higher efficiency than the PCR assay in the detection of the shrimp sample.
4. Optimized Vibrio spp. universal LAMP detection system need incubated at62℃for60min. Universal LAMP with a highly specificity for Vibrio spp. detection. The detection sensitivity was100times higher than the PCR method. The universal LAMP method showed very good detection ability when used in the detection of Vibrio from artificial infected aquatic animals.
The results showed that the LAMP method is a simple, sensitivity and high specificity for the rapid pathogen detection. With no need for special equipment, it suitable for rapid detection kit development. LAMP method was a significant potential tool for high throughout and fast diagnosis against aquatic pathogens in the field.
本研究新建了环介导等温扩增方法分别快速检测对虾白斑综合征病毒、皮下及造血组织坏死病毒和弧菌。首次进行了白斑综合征病毒和皮下及造血组织坏死病毒进行同时检测的研究,以及首次对某一类特定病原体进行通用LAMP检测研究,研究了LAMP对弧菌属细菌的通用性检测。设计不同病原相应的检测引物,分别对其反应条件(镁离子浓度、甜菜碱浓度、反应时间、反应温度等)进行了优化,并检测了其特异性及灵敏度。首次将dUTP-UNG酶(尿嘧啶-N-糖基化酶)技术引入LAMP检测中,克服了LAMP反应极易产生假阳性的缺点,并对dUTP对dTTP的替代率、替代对反应灵敏度的影响进行了研究。并将建立好的LAMP检测方法用于实际水产动物样品的病原检测。具体结论如下：
1、优化后的白斑综合征病毒LAMP反应体系在64℃下45min即能有理想扩增效果。加入SYBR Green I后,能用肉眼进行结果的判断。使用常见细菌和对虾病毒做对照,显示LAMP反应对WSSV检测具有很好的特异性。灵敏度达到100copies/μl,比巢式PCR高100倍。反应中使用dUTP替代部分dTTP,结合UNG酶,可以避免LAMP的产物遗留污染的问题,但替代率高于50%后,显著抑制反应的扩增效率。在对虾样品的WSSV检测中,LAMP方法表现出比巢式PCR更好的检测灵敏度和更快速的检测流程。
2、皮下及造血组织坏死病毒LAMP反应体系在65℃反应60mmin完成扩增反应。产物经荧光染色后发生颜色变化,产物检测方便。对扩增产物进行酶切得到与预期相吻合的片段。50%的dUTP替代和JNG酶的使用能避免假阳性的发生,但会使反应灵敏度下降10倍。LAMP反应对IHHNV具有较好的特异性。其LAMP灵敏度较普通PCR高1000倍。对实际对虾样品的IHHNV检测中,LAMP方法于PCR方法结果一致,但表现出更高的效率和更加直观准确的结果。
3、优化后的WSSV和IHHNV多重LAMP反应体系需在65℃反应60min。产物经过酶切得到的与预期大小相吻合的条带。多重LAMP具有较好的特异性和较高的灵敏度,比巢式PCR高两个数量级,较普通PCR高三个数量级。在实际对虾样品的检测中,多重LAMP检测较PCR检测具有更高的病毒检出率和更高的检测效率。
4、优化后的弧菌属细菌通用LAMP检测的反应体系需在62℃反应60min。通用LAMP检测对弧菌属细菌具有高度的特异性。检测灵敏度较普通PCR方法高100倍。将通用LAMP检测方法应用于人工感染弧菌的水产动物检测上,表现了很好的检测效果。
检验结果表明,LAMP检测方法是一种操作简单、灵敏度和特异性较高的快速检测手段；无需特殊仪器设备,非常适合基层养殖场和防疫部门开展实地检测,同时也适合开发为快速检测试剂盒,在水产病原的实地快速高诊断方面具有重大的开发潜力。
With the scale expansion of aquaculture and degradation of aquatic environment, diseases of aquatic animals has become more and more serious, which is one of the bottlenecks of the aquaculture development. Rapid diagnosis of aquatic pathogens was of great significance for breeding, disease prevention, import and export quarantine, aquatic food safety, etc. Since their individual deficiency, most of the conventional pathogen detection techniques are not suitable for rapid field detection. Loop-mediated isothermal amplification (LAMP) is a new DNA amplification method. It relies on the automatic cycle of the strand displacement reaction. With a set of primers targeting six specific regions and DNA polymerase with strand displacement, it can amplify target nucleic acids from a few copies to109copies within1h. High specificity, sensitivity and efficiency, with easy protocol, make LAMP technique suitable for rapid field detection.
In this study, LAMP methods for rapid detection of white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Vibrios were developed. Multiplex LAMP for detecting WSSV and IHHNV at the same time and universal LAMP for detecting a group of specific pathogens were developed for the first time. The universal LAMP was used to detect Vibrio spp. Different primers were designed to targeting different pathogens DNA sequences. LAMP reaction conditions were optimized, including concentration of magnesium ions, the betaine concentration, reaction time, reaction temperature, etc. And the specificity and sensitivity were tested. DUTP-UNG (uracil-N-glycosylase) system was used to overcome carry-over contamination of LAMP for the first time. The replacement rate of dUTP to dTTP and the influence of the replacement to LAMP sensitivity were studied. LAMP detection systems were used for aquatic animal samples detection. Specific conclusions are as follows:
1. Incubating the optimized LAMP reaction for WSSV at64℃for45min can have the effect amplification. By adding SYBR Green I, the results can be judged by naked eyes. Using common bacteria and shrimp viruses as controls, LAMP method has good specificity WSSV detection. Sensitivity of LAMP was100copies/μl, which was100times higher than the nested PCR. DUTP replacing part of dTTP in the reaction mixture, combining the UNG enzyme, LAMP carry-over contamination can be prevented. But when the replacement rate was higher than50%, amplification efficiency was significantly inhibited. The LAMP method has higher sensitivity and faster detection process than nested PCR in shrimp samples detection against WSSV.
2. LAMP reaction system for IHHNV amplified properly at65℃for60min. Reaction tube color changed after staining, that makes it convenience for judgment. Expected fragments appeared after amplicons were digested by restriction enzyme. Fifty percent of the dUTP substitution and the using of the UNG can avoid the false positives, but that made the sensitivity decrease by about10times. LAMP detection showed good specificity for IHHNV. Its sensitivity was1000times higher than common PCR. In shrimp samples IHHNV detection, the LAMP method was consistent with PCR, but showed a higher efficiency and more intuitive results.
3. Optimized mLAMP system for WSSV and IHHNV detection was incubated at65℃for60min to complete the reaction. After digestion, fragments of amplicons were consistent with the expectation. Multiplex LAMP has high specificity and sensitivity, which was100times higher than nested PCR and1000times higher than the conventional PCR, respectively. Multiplex LAMP detection showed higher detection rates and higher efficiency than the PCR assay in the detection of the shrimp sample.
4. Optimized Vibrio spp. universal LAMP detection system need incubated at62℃for60min. Universal LAMP with a highly specificity for Vibrio spp. detection. The detection sensitivity was100times higher than the PCR method. The universal LAMP method showed very good detection ability when used in the detection of Vibrio from artificial infected aquatic animals.
The results showed that the LAMP method is a simple, sensitivity and high specificity for the rapid pathogen detection. With no need for special equipment, it suitable for rapid detection kit development. LAMP method was a significant potential tool for high throughout and fast diagnosis against aquatic pathogens in the field.
引文
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