FTA卡保存对虾传染性皮下和造血组织坏死病毒DNA洗脱方法的优化

Optimization of DNA elution method of infectious hypodermic and hematopoietic necrosis virus preserved by FTA card

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

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 (Tris-EDTA)缓冲液及去离子水为基础洗脱液，设计7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4 mm2的FTA卡上，点样体积为2.5 μL，用洗脱液作为模板时，最低点膜核酸浓度需要1.47×104 copies/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50 μL TE缓冲液于95 ℃下浸洗5 min；用膜片做模板，点膜核酸浓度需要1.82×103 copies/μL以上，室温(20~25 ℃)条件下，用FTA纯化试剂洗脱3次，再用TE缓冲液洗脱2次，洗脱时间均为5 min，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类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数据。

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 may fulfil the requirements of long-distance and cross-border sample transportation. The FTA card can store and transport tissue, nucleic acid, and other sample types at room temperature (20–25 ℃). Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods, eliminating tedious processes, saving time, and ensuring sample quality. It is widely used in the human and animal medicine field. 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) and the shrimp Enterozoon hepatopoaei (EHP): However, 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. The infectious hypodermal and haematopoietic necrosis virus (IHHNV) is an important shrimp pathogen and severely impacts the shrimp culture industry. It was first found in Hawaii, United States, in 1981 and then spread to several countries, including to Australia, Singapore, Malaysia, South Korea, Brazil, and China. The IHHNV infecting Penaeus vannamei does not cause high mortality, but growth would become slow and deformed, resulting in great economic losses. Early detection and prevention management are particularly important in current situations, which lack effective control measures for the disease. Several IHHNV detection methods have been established that use molecular biological methods, including conventional polymerase chain reaction (PCR) and real-time PCR (these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health). Nucleic acid extraction by the above methods meets the requirements for samples, usually frozen, ethanol, or other nucleic acid preservation reagents. Low temperature preservation conditions and composition restrictions of preservation solutions present certain difficulties in disease investigation, surveillance, and monitoring of shrimp farming. It is particularly important to address this dilemma. 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 cards as the preservation medium, and designed seven kinds of FTA cards with attached DNA elution methods based on the FTA purification reagent, TE buffer, and deionized water. We evaluated the elution and separation effects of different nucleic acids and the minimum amount of dot FTA card nucleic acids through real-time PCR detection. The appropriate solution was spotted onto FTA cards according to the manufacturer´s protocol, labeled, and air-dried for 1 day at room temperature. The result shows that on the 4 mm2 FTA card, the sample volume was 2.5 μL. When the eluent is used as the template, the minimum FTA card nucleic acid concentration needs to be 1.47×104 copies/μL above the best detection sensitivity, and 100% detection rate can be obtained by washing the FTA card with 50 μL TE buffer solution at 95 ℃ for 5 min. Using the FTA card as the template, the nucleic acid concentration of the dot FTA card needs to be above 1.82×103 copies/μL, eluted with FTA purified reagent thrice at room temperature, and then eluted with TE buffer twice. Each elution time is 5 min as this can obtain the best detection sensitivity and demonstrates 100% accuracy. The elution effect of the above two schemes was better than that of the other five schemes. The nucleic acids of WSSV, EHP, decapod iridescent virus 1, covert mobility Noda virus, and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis were preserved using FTA card to test the efficiency of the established elution method. It is assumed that this method is universal for the elution of other shrimp pathogenic nucleic acids. At present, 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 nucleic acid, separation methods, and detection effect. This study shows that FTA cards used to preserve pathogenic nucleic acid requires a specific amount of nucleic acid in the sample and 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 cards. The application of this technology has potential use as storage and transport strategy for surveillance programs and can 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.