Abstract
Enterocytozoon hepatopenaei (EHP) is a pathogen in the pancreatic tissue of prawn, as reported in recent years. Enterosporidiosis caused by EHP in Penaeus genus is spreading widely, which seriously threatens the sustainable development of shrimp aquaculture in the world. Empolying the DNA binding dye of SYTO-16, a real-time quantitative loop-mediated isothermal amplification (LAMP) method has been established herein to detect EHP. The primer sequences used in the LAMP reaction were according to the SSU rRNA gene. The LAMP assay has reached a sensitivity of 101 copies/µL and no cross-reaction with other aquatic pathogens. Compared with normal PCR, the efficiency of the LAMP technique is more sensitive, which has a shorter detection time. The use of fluorescent nucleic acid dye (SYTO-16) reveals a more satisfactory performance relative to calcein. The quantitative LAMP assay can be considered as a valid tool for rapid detection of microsporidian in prawns. Our study provides a scientific basis to follow the effect of the pathogen infection on growth of cultured penaeid shrimp.
Highlights
In recent years, infection of Enterocytozoon hepatopenaei (EHP) in Penaeus vannamei had been widely spread in the Asia-Pacific region[1]
The established quantitative loop-mediated isothermal amplification (LAMP) assay by using SYTO-16 appears to be an accurate and sensitive method for rapid detection of the pathogen in shrimp and for early diagnosis and intervention in shrimp aquaculture
The fluorescence-quantitative LAMP assay was conducted under isothermal conditions between 60 °C and 65 °C
Summary
Infection of Enterocytozoon hepatopenaei (EHP) in Penaeus vannamei had been widely spread in the Asia-Pacific region[1]. Over the past several decades, several reliable and powerful molecular diagnostic techniques such as polymerase chain reaction (PCR)[2], nested PCR4 and quantitative PCR (qPCR)[5] have developed to trace the pathogen These methods require fully equipped laboratories with good infrastructure, reliable electrical supply, and highly trained staffs. The loop-mediated isothermal amplification (LAMP) assay was an excellent diagnostic tool because of its simplicity, cost-effectiveness, high efficiency, and specificity[7] This method needed a four-primer set, designed to recognize six distinct regions on the target gene, and required the enzyme Bst polymerase which had strand displacement activity. The established quantitative LAMP assay by using SYTO-16 appears to be an accurate and sensitive method for rapid detection of the pathogen in shrimp and for early diagnosis and intervention in shrimp aquaculture
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