Establishment Of Duplex QPCR/ddPCR For Detection Of Enterocytozoon Hepatopenaei And Acute Hepatopancreatic Necrosis Disease In Shrimp

Enterocytozoon hepatopenaei(EHP)and acute hepatopancreatic necrosis disease(AHPND)are two of the diseases that frequently infect farmed shrimp in recent years,causing great economic losses to aquaculture industry worldwide.It was reported that shrimp infected with EHP will increase the susceptibility to AHPND,and there have been cases of EHP-AHPND mixed infection.In order to prevent and control the spread and outbreak of EHP and AHPND,this study established duplex qPCR and duplex dd PCR detection method based on the SWP1gene of EHP and the pir A gene of AHPND that can simultaneously detect these two pathogens.After testing the diagnostic feasibility of single PCR,optimized the ratio of primer concentration to probe concentration,annealing temperature and et al to determined the best reaction system for duplex PCR,and then performed specificity,sensitivity,repeatability and et al.The results are as follows:1.When the ratio of primer concentration to probe concentration for two target were 400nM:200 nM,and the annealing temperature was 60?,duplex qPCR had the best amplification effect.This method can specifically detect EHP and AHPND,with the lowest detection limits of3.3 copies/?L and 3.2×10^-1copies/?L respectively,and the correlation coefficients(R²)of the standard curve are 0.9916 and 0.9832.The maximum CV(%)value of repeated experiments within and between groups was less than 0.1.These results showed that the duplex qPCR for EHP and AHPND has strong stability,good detection effect and repeatability.2.When the ratio of primer concentration to probe concentration was 500 nM:250 nM,and the annealing temperature was 55?,the duplex dd PCR showed the best amplification effect with good specificity.It could detect EHP recombinant plasmid as low as 3.3×10^-1copies/?L and AHPND recombinant plasmid as low as 3.2×10^-2copies/?L.The correlation coefficients(R²)of the duplex dd PCR standard curves for EHP and AHPND were 0.9663 and 0.9816,respectively;the maximum CV(%)values of repeated experiments within and between groups were both less than 0.6.In addition,this study further verified that the supercoiled structure of plasmid DNA was an important factor affecting the amplification efficiency of dd PCR.The above results indicated that the duplex dd PCR established in this study has stable system and excellent detection accuracy for EHP and AHPND,which can be used as an effective means for quantitative detection of these two pathogens.3.72 suspected diseased shrimps were used for the practical application of duplex qPCR and duplex dd PCR.For EHP,the detection rates of duplex qPCR and duplex dd PCR were 23.6%(17/72)and 27.8%(20/72)respectively;for AHPND,the detection rates of duplex qPCR and duplex dd PCR were 26.4%(19/72)and 27.8%(20/72),respectively.With the verification of nested PCR and Sanger sequencing,the duplex dd PCR had 100%accuracy in the pathogens detection;and the diagnostic sensitivity and diagnostic specificity of duplex qPCR were both greater than 80%.The quantitative results of samples analyzed by Pearson's correlation coefficient showed that the established duplex qPCR and duplex dd PCR have good consistency in the quantitative detection for practical sample.In summary,comprehensive evaluation and analysis of various experiments showed that the EHP/AHPND duplex qPCR and duplex dd PCR detection methods established in this study were convenient,rapid,high sensitivity and excellent quantitative effect.They were effective means for the diagnosis of EHP and AHPND,and could provide certain technical support for the research and control of shrimp disease.