| Enterocytozoon hepatopenaei(EHP) is a kind of pathogen parasitic on shrimp hepatopancreas tissue, which is founded in recent years. And EHP was discovered and got its name from the slow-growing giant black tiger shrimp Penaeus monodon from Thailand in 2009. Although EHP doesn’t appear to cause mortality, it will reduce the growth of the shrimp. EHP was neglected because of the overwhelming focus on early mortality syndrome(EMS) and acute hepatopancreatic necrosis disease(AHPND). In this case, EHP has been spread more widely. There is information indicating that EHP has been quarantined in China, Indonesia, Malaysia, Vietnam, India and Thailand. This thesis will concentrate on issues below: the detection of the real-time fluorescence quantitative, challenge tests, the effect of the EHP quantity on shrimp.A pair of specific primers has been designed according to the EHP SSU r DNA sequences published in Gen Bank. The primers have nice property and high sensitivity which is resulted from the study of its characteristics. To construct recombinant plasmid by using p MD18-T containing SSU r DNA sequences as experimental standards. After several optimization of the reaction system, the q PCR method is successfully established to test the EHP SYBR GreenⅠ. The results show that the method of constructing has a good linear relationship, amplification effect reaches best when Tm is 60℃, melting curve only gets a single specific peak, The cycles of amplification threshold(Ct) has a wild linear range responding the logarithmic starting quantity of the template [log(Sq)] between 8.3 × 101 — 8.3 × 108 copies/μL matching a standard curve of Ct =-3.369 log(Sq) + 39.364, the correlation coefficient is R2=0.992, the amplification efficiency is 98.5%. By examing the growth of the 31 vannamei samples collected from Hainan in uneven size with the method of this experiment, test results shows the positive rate is 68%--higher than conventional PCR, additionally, melting curve analysis shows a good specificity, which all means the method has good applicability. SYBR GreenⅠq PCR established in this study is specific, sensitive, rapid and quantitative, and it can offer a reference for quantitative research of EHP.Challenge test was carried out using normal P. vannamei juveniles and Artemia juveniles were fed with frozen and fresh material hepatopancreatic tissue.A week later, PCR examination turn out normal P. vannamei juveniles were infected EHP. The experiment shows that the enterocytozoon hepatopenaei has the ability to resist severe environment and still have infectivity, which means it is difficult to prevent and treat the enterocytozoon hepatopenaei infection.During the epidemiological investigation process in EHP,the L. vannamei samples were collected from Jimo Shandong, Ganyu Jiangsu and Danzhou Hannan where the samples are growing slowly and the sizes are very different. The length of the samples are measured and it is suspicious that the EHP is the cause of slow-growth and size differences. And then use the q PCR detection method to get the effect of EHP on these samples. Meanwhile, PCR test on the other four pathogens as WSSV, IHHNV, CMNV, AHPND. And the result shows that the detection rates is relatively low. EHP in Danzhou Hainan is 68%, and all positive in Ganyu Jinagsu and Jimo Shandong. Hepatopancreatic DNA(Hp DNA) samples extracted from 94 shrimp L. vannamei collected in farms in Jiangsu, Hainan, and Shandong Provinces were individually detected by the q PCR. The results showed that the EHP loads in the hepatopancreas negatively correlate with the shrimp growth rate. The EHP load above 103 copies/(ng Hp DNA) represents a high risk level. The q PCR method developed in this study has the advantages of specificity, sensitivity, rapidness and quantifiability. This method and the application data can provide a useful tools and reference for the prevention and control of shrimp microsporidian E. hepatopenaei. |
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