Rapid Preparation Reagent for RPA-LFD Detection of DNA Samples from the Shrimp Hepatopancreas

Abstract

This study is committed to developing and optimizing a rapid nucleic acid preparation reagent (nucleic acid release agent) that is suitable for recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) technology to detect DNA nucleic acid samples in shrimp hepatopancreas to realize on-site rapid detection and remove the cumbersome and time-consuming steps of conventional nucleic acid sample extraction.The optimum compositions of nucleic acid release agent are 20–100 mmol/L Tris-HCl, 50–250 mmol/L KCl, 0.01%–0.10% (W/V) lithium dodecyl sulfate (LDS), 0.5%–2.0% (V/V) Triton X-100, 1–5 mmol/L ethylenediaminetetraacetic acid disodium salt (EGTA2Na), 0.5–5.0 mmol/L bovine serum albumin (BSA), 1–5 mg/ml gelatin, 0.01%–0.10% (W/V) trehalose, and 1%–5% (W/V) betaine. The proportion of each component of nucleic acid release agent was optimized using positive and negative samples of shrimp Enterocytozoon hepatopenaei (EHP). The RPA-LFD reaction initially involved collecting shrimp hepatopancreas (the size of a mung bean), adding 100 μL nucleic acid release agent, heating for 3 min at 100 ℃ and using the supernatant for analysis. The optimal ratio of each component of nucleic acid releaser was determined to be 100 mmol/L Tris-HCl, 100 mmol/L KCl, 0.02% LDS, 0.5% Triton X-100, 1 mmol/L EGTA2Na, 0.05% trehalose, 1 mg/mL gelatin, 0.5 mmol/L BSA, and 2% betaine. The positive and negative samples of acute hepatopancreatic necrosis disease (AHPND) were used as detection templates to verify the optimized nucleic acid release agent. The optimized nucleic acid release agent could be used to prepare nucleic acid samples, and the prepared samples were available for RPA-LFD method testing. The optimized nucleic acid release agent in this study can be used to prepare DNA nucleic acid samples of the shrimp hepatopancreas for RPA-LFD detection. It avoids the tedious and time-consuming preparation steps of conventional DNA nucleic acid samples and greatly improves the efficiency of nucleic acid level pathogen detection.Disease is the bottleneck that restricts the green development of the shrimp breeding industry, which can cause huge annual economic losses. Detection and monitoring of shrimp related pathogens are effective to solve this problem. Enterocytozoon hepatopopenaei (EHP) and Vibrio parahaemolyticus (VP) are two common pathogenic microorganisms in shrimp culturing that cause AHPND. E. hepatopopenaei is an obligate intracellular parasite that mainly parasitizes in the hepatopancreas of shrimp. Severe EHP infections lead to atrophy of the hepatopancreas of shrimp. E. hepatopopenaei has a wide range of hosts, including Penaeus vannamei, P. monodon, and Palaemon carincauda. AHPND is an aquatic disease caused by Vibrio parahemolvticus with the virulent plasmid PirA/B. It was first found in China and Vietnam in 2010, then in Malaysia, Thailand, Mexico, the Philippines, and other countries. P. monodon, P. vannamei, P. chinensis, and P. japonicus are susceptible hosts. The distribution of pathogenic vibrio can be detected in the hepatopancreas tissue of diseased shrimp, and it can cause hepatopancreas atrophy. Broadly, most shrimp pathogens (such as DIV1, WSSV, TSV, and IHHNV) infect the hepatopancreas of shrimp. DIV1 infections cause pale atrophy of the hepatopancreas. Histopathological examination identified eosinophils and dark eosinophilic inclusions in the cytoplasm of the hepatopancreas of shrimp infected with DIV1. Some of them were coated or contained slight basophilic staining, and hemocytes were pyknotic. WSSV and TSV can cause pale atrophy symptoms of the hepatopancreas, while IHHNV can also cause hepatopancreas enlargement of shrimp. Therefore, the nucleic acid samples of the hepatopancreas of shrimp can be widely used as templates for detection of most shrimp pathogens.Isothermal nucleic acid amplification technology removes the dependence on conventional nucleic acid temperature change amplification using the PCR amplification instrument, and is widely used for rapid detection. Currently, isothermal amplification technology covers loop mediated isothermal amplification (LAMP), strand displacement amplification (SDA) and recombinase polymerase amplification (RPA), and so on. Among them, RPA technology was developed in recent years using recombinases to promote the insertion and binding of oligonucleotide primers in DNA double strand complementary sequences, and realize exponential amplification of specific DNA regions under the action of Bsu DNA polymerase. It has a short reaction time, strong specificity, high sensitivity, and operates at a constant temperature of 37–42 ℃. It is suitable for on-site detection and analysis. The combination of RPA with the lateral flow dipstick (LFD) to read the detection results is simple to operate, fast, with high specificity and sensitivity. It is a new method suitable for rapid detection in the field. The combination of RPA-LFD technology and a simplified nucleic acid extraction method can help to realize portable on-site rapid nucleic acid detection.

Keywords

• nucleic acid releasing reagent
• recombinase polymerase amplification
• lateral flow dipstick
• shrimp hepatopancreas
• pathogen rapid detection