Optimization Of Multiple Nested PCR (MNPCR) Detection Method For Five Shrimp Pathogens

With the large-scale development of the white shrimp Litopenaeus vannamei farming,the threat of its disease is increasing.It is of great significance to timely monitor the pathogens status and scientifically apply ecological prevention and control technology to ensure the production efficiency of shrimp,thereby promoting the healthy development of the industry.This study aims to establish a detection technology targeting simultaneously for five major common pathogens of white shrimp,such as Shrimp hemocyte iridescent virus(SHIV),Enterocytozoon hepatopenaei(EHP),Vibrio parahaemolyticus for Acute hepatopancreas necrosis disease(VPAHPND),Infec-tious hypodermal and hernatopoietic necrosis virus(IHHNV)and White spot syndrome virus(WSSV).Furthermore,it can provide technical supports to develop an ecological control technology on shrimp diseases.In this study,PCR detection technology for single pathogen was firstly used to investigate and analyze infection situation of shrimp pathogens(including SHIV,EHP and VPAHPND)in main aquaculture areas of Maoming and Shanwei from 2017 to 2018.The results found that SHIV,EHP and VPAHPND were all detected in farmed shrimp.Among them higher positive rates of 23.81%and 23.13%were detected for SHIV and EHP,and relatively low rate of 3.40%was detected for VPAHPND.SHIV,EHP and VPAHPND were also detected in pond water and source water.Among them,positive rates of SHIV were 30.43%and 44.00%,positive rates of EHP were 47.83%and 36.00%,and positive rates of VPAHPND were 4.35%and 4.00%,respectively.Occasionally,a certain amount of VPAHPND could be detected in shrimp seedlings.It was also found to be positive for EHP and VPAHPND in food organisms of Artemia for shrimp larvas.According to the above results,there pathogens of SHIV,EHP and VPAHPND plus other two traditional common pathogens of WSSV and IHHNV were selected as the detection targets.The specific nested PCR primers for SHIV,EHP,VPAHPND,WSSV and IHHNV were designed and optimized respectively.The multiple nested PCR(MNPCR)technology that can simultaneously detect the five pathogens was established and optimized by referring to the positive plasmids of the five pathogens constructed.The results showed that the amplification product fragment sizes of SHIV,EHP,VPAHPND,WSSV and IHHNV were 129 bp,176 bp,402 bp,230 bp and 294 bp,respectively.The minimum detection levels were 1×102copies/μL for IHHNV,and 1×101copies/μL for VPAHPND,WSSV,EHP and SHIV.Subsequently,shrimp samples were collected from ponds without disease outbreak in main coastal aquaculture areas of Guangdong province.The results showed that the positive rates of SHIV,EHP and WSSV were 8.02%,54.01%and 15.51%,respectively.Compared to shrimp samples from ponds with diseases in Shanwei,the positive rates of them were 100%,77.78%and 44.44%respectively.It can be seen that this MNPCR detection method for shrimp pathogens is feasible.It can provided real-time dada of pathogens for making plans to shrimp disease prevention and control.