Shrimp disease detected sharp weapon!It is convenient and fast to detect EHP infection

EHP (Enterocytozoon Hepatopenaei) is an intracellular parasite that infects eukaryotes and, if it infects prawn, can cause growth retardation related diseases in prawn.One, EHP in prawn culture, great harm.They are parasites that destroy epithelial cells in the hepatopancreatic ducts and prevent shrimp from absorbing nutrients. Typical symptoms of EHP infection in shrimp are slow growth, stunted growth, soft shell, and white stool.In Asian countries such as India, Thailand and China, EHP has been reported to reduce annual shrimp yields by 10-20%, causing significant economic losses.EHP was first reported in Thailand in 2009, according to the records.As prawns are infected with EHP, there are no symptoms or any specific clinical symptoms.Therefore, diagnostic tests are considered to be the most effective screening method.For example, there are many ways to detect EHP in shrimp diseases by conventional PCR, qPCR (real-time fluorescence quantitative PCR), or LAMP (loop-mediated isothermal amplification reaction).All of these tests are a common technique for amplifying DNA from diseased shrimp samples, which is now widely used to identify diseases caused by microorganisms.However, these methods require high technology, advanced equipment, expensive testing costs, and a lot of time.LAMP method takes only 45 minutes to react, but it still needs advanced high-precision temperature controller.EHP spores in (A) HP stained cells, (B) PART of HP stained tissues.Recently, RPA (Recombinase polymerase amplification) technology is essentially based on the activity of recombinase and polymerase (enzymes synthesizing polymer chains or nucleic acids) to amplify nucleic acids (including DNA and RNA), which has been applied and evaluated as having great potential in the diagnosis of parasites and viral diseases.The key to RPA technology is that the amplification step of traditional PCR (amplification with template DNA) is replaced by recombinant enzymes and SSB molecules. Therefore, RPA can be isothermal amplification (or thermostatic amplification) at 37-42ºC and sample identification time is only about 5-20 minutes.Single-stranded binding protein (SSB) molecules are made up of protein molecules that keep the two single strands of DNA from sticking together after they split, allowing the enzyme to work.It is well known that the three stages of PCR test require three temperature components: denaturation phase, where the temperature rises to 94ºC, causing the DNA double strand to unravel;The next stage is the pairing stage, where the temperature is lowered to about 55 ºC-65 ºC for additional pairing of primers;Finally, the elongation stage, at which the temperature needs to reach 72℃, the Tag enzyme can play its role and combine into a complete complementary chain.This cycle repeats about 25-40 times, depending on the type of reaction.Researchers have attempted to use RPA technology to detect EHP disease in prawn, simplifying the process without the need for temperature control.However, the analysis of electrochemical gain products on gels is only allowed in the laboratory and is difficult to apply in practice, and sensitivity is limited by gel imaging tools.A recent study used fluorescence signal reading and RT-RPA assay (real-time RPA) to detect EHP disease in shrimp.The test uses SWP gene encoding EHP spore wall protein, which has a higher molecular diagnosis than SSU rRNA gene, and the results of previous studies have confirmed the good specificity of this gene for EHP detection.The RT-RPA test showed good sensitivity, comparable to nested PCR (which has a very high sensitivity), and much better than RPA assay using gel electrophoresis analysis.The sensitivity of RT-RPA was nearly 60 times higher than that of RPA assays analyzed by gel electrophoresis.In addition, rT-RPA results of the samples were 100% similar to nested PCR, showing good reliability.With fluorescent probes, results can be confirmed by visualization within 15 minutes.Small, battery-powered fluorescent tube scanners can be used to signal in situ RT-RPA tests.With a low reliance on equipment and power, THE RT-RPA can be easily assembled into a portable, convenient laboratory suitcase.RPA has been used to diagnose viral pathogens such as WSSV, IHHNV and SHIV.Conclusion: The RT-RPA test kit combined with fluorescent probe readings can quickly detect EHP infection in shrimp.It can be used as an effective and reliable in situ detection tool to help control EHP infection in shrimp farms.

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