Optimization of DNA Elution Method of Infectious Hypodermic and Hematopoietic Necrosis Virus Preserved by FTA Card

Abstract

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 Enterocytozoon hepatopenaei (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.

Keywords

• ihhnv
• flinders technology associates cards
• elution method
• optimization