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| FTA卡保存对虾传染性皮下和造血组织坏死病毒(IHHNV)DNA洗脱方法的优化 |
| Optimization of DNA Elution Method of Infectious Hypodermic and Hematopoietic Necrosis Virus (IHHNV) Preserved by FTA Card |
| 投稿时间：2022-12-16 修订日期：2023-02-01 |
| DOI： |
| 中文关键词: IHHNV FTA卡 洗脱方法 优化 |
| 英文关键词: IHHNV Flinders Technology Associates Cards Elution method optimization |
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| 中文摘要: |
| 传染性皮下和造血组织坏死病毒(Infectious hypodermal and haematopoietic necrosis virus, IHHNV)是危害虾类健康养殖的重要病原,为寻找一种快捷保存及分离IHHNV DNA的方法,为后续研究提供完整的核酸材料,选用FTA(Flinders Technology Associates)卡为保存介质,以FTA纯化试剂、TE缓冲液及去离子水为基础洗脱液,设计了七种FTA卡黏附DNA洗脱方法,通过荧光定量PCR检测方法检验不同核酸洗脱分离效果及最低的点膜核酸量。结果显示,于4mm2的FTA卡上,点样体积2.5μL,用洗脱液作为模板时,最低点膜核酸浓度需要1.47×104copies/μL以上,可获得最佳的检测灵敏度和100%检出率的洗膜方法为50uLTE缓冲液于95℃下浸洗5min；用膜片做模板,点膜核酸浓度需要1.82×103copies/μL以上,室温下用FTA纯化试剂洗脱3次,再用TE缓冲液洗脱2次,各洗脱时间均为5min,可获得最佳的检测灵敏度和100%的准确度。以FTA卡保存对虾白斑综合征病毒(white spot syndrome virus,WSSV)、虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)、虾十足目虹彩病毒1(Decapod iridescent virus 1, DIV1)、偷死野田村病毒(Covert mortality noda virus,CMNV)、致急性肝胰腺坏死副溶血弧菌(Vibrio parahaemolyticus,VpAHPND)核酸,测试所建洗脱方法的效果,证实了该方法对其他虾病原核酸的洗脱具有通用性。该研究给出了FTA卡保存和洗脱IHHNV DNA的适用性方案,为野外对虾样品采集、病毒核酸样品跨区域传递的保存和运输条件提供了科学依据。 |
| 英文摘要: |
| Flinders Technology Associates (FTA) card (Whatman?) is a paper-based matrix designed to fix, purify and store genetic material from various biological sources. It can conveniently and quickly preserve nucleic acids and be required the purpose of long-distance and cross-border transportation of samples. FTA card can store and transport tissue, nucleic acid and other types samples at room temperature. It could be directly extracted nucleic acid for detection and be sent by express as an ordinary parcel without being treated as dangerous and special goods, eliminating the tedious process, saving time and ensuring sample quality. It is widely used in human and animal field of medicine. And it has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids. In terms of aquatic animals, the FTA card has been used by researchers to store white spot syndrome virus (WSSV), shrimp enterozoon hepatopoaei (EHP), etc., but there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card, which affects the application of the FTA card. Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is important disease pathogen of shrimp and seriously impacting shrimp culture industry in the world. It was first found in Hawaii in the United States in 1981, and then spread to many countries and regions around the world, including Australia, Singapore, Malaysia, South Korea, Brazil, China, etc. The infection of Litopenaeus vannamei with IHHNV will not cause high mortality, but the growth to be slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in the current situations which has lack of effective control measures for the disease. There are several IHHNV detection methods have been established in which molecular biological methods are widely used including conventional PCR method and real-time PCR method those are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health (WOAH). Nucleic acid extraction by the above methods shall meet the requirements for samples, usually frozen, ethanol or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions bring certain difficulties to disease investigation, surveillance and monitoring of shrimp farming. It is particularly important to break through this dilemma. In order to find a fast method for the preservation and separation of IHHNV DNA and provide complete nucleic acid materials for subsequent research, we selected FTA card as the preservation medium, and designed seven kinds of FTA card attached DNA elution methods based on FTA purification reagent, TE buffer and deionized water. We tested the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. TheSappropriate solution was spotted onto FTA cards according to the manufacturer’s protocol, labeled and air-dried for one day at room temperature. The result shows that on the 4mm2 FTA card, the sample volume is 2.5μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104copies/μL above, the best detection sensitivity and 100% detection rate can be obtained by washing the FTA card with 50uL TE buffer solution at 95℃ for 5min; Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs 1.82×103copies/μL above, elute with FTA purified reagent for 3 times at room temperature, and then elute with TE buffer for 2 times. Each elution time is 5min, which can obtain the best detection sensitivity and 100% accuracy. The elution effect of the above two scheme were better than that of the other five schemes. Preserve the nucleic acids of white spot syndrome virus (WSSV), Enterocytozoon hepatopoaei (EHP), Decapod iridescent virus 1, (DIV1), Covert mobility noda virus (CMNV), and Vibrio parahaemolyticus (VpAHPND) causing acute hepatopancreatic necrosis with FTA card, and test the effect of the established elution method. It is proved that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, the research on the application of FTA card is mostly seen in the nucleic acid effect of its preservation and transportation of tissue samples. There are few reports on the relationship between the amount of preserved samples, separation methods and detection effect. This study shows that the FTA card used for preservation of shrimp pathogenic nucleic acid not only requires the amount of nucleic acid in the sample, but also directly affects the detection results of the sample with different FTA card elution methods. This study provides a feasible scheme for the preservation and elution of IHHNV DNA with FTA card. The application of this technology has potential use as storage and transport strategy for surveillance programmes and enhance biosecurity in shrimp culture, which provides a scientific basis for the preservation and transportation conditions for the collection of wild shrimp samples and the regional transmission of viral nucleic acid samples. |
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