【作者】 肖广侠；

【导师】 孔杰；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 白斑综合征病毒（White spot syndrome virus, WSSV）是造成中国对虾暴发性流行病的主要病原,迄今没有能有效控制WSSV疫情的技术措施。为预防和减少WSSV对中国对虾造成的损失,本文比较了3种PCR方法检测WSSV灵敏度的差异,对中国对虾WSSV携带量与繁殖、存活及育种值进行相关性分析,取得主要结果如下:1、三种PCR方法检测白斑综合征病毒（WSSV）的灵敏度比较分别利用TaqMan实时定量PCR、世界动物卫生组织（Office International Des Epizooties, OIE）公布的巢式PCR引物（简称OIE巢式PCR）及本研究室设计的引物（简称GB巢式PCR）和OIE巢式PCR及GB巢式PCR对应的一步法PCR等三种方法对不同白斑综合征病毒（White spot syndrome virus,WSSV）含量的中国对虾样品进行灵敏度比较。结果显示,当使用已知病毒含量的标准品进行检测时,TaqMan实时定量PCR方法可以检测到10个WSSV copies,OIE巢式PCR与GB巢式PCR方法分别可检测到10⁴和10³个WSSV copies。分别使用OIE巢式PCR的外引物和内引物扩增时,可检测到5×10⁴和2.5×10⁴个WSSV copies;分别使用GB巢式PCR的外引物和内引物扩增时,可检测到10⁴和5×10³个WSSV copies。使用上述PCR方法对44份未知WSSV含量的样品进行验证,定量PCR方法检测阳性率为84.09%,OIE巢式PCR与GB巢式PCR方法检测的阳性率分别为18.18%和27.27%。分别使用OIE巢式PCR的外引物和内引物扩增检测的阳性率均为15.91%,分别使用GB巢式PCR的外引物和内引物扩增检测的阳性率分别为18.18%和20.45%。根据以上结果,这三种方法检测WSSV的灵敏度由高到低依次为:TaqMan实时定量PCR、巢式PCR、一步法PCR。2、不同饵料组合对越冬对虾WSSV携带量及繁殖特征的影响对越冬对虾使用的2种饵料菲律宾蛤仔和沙蚕进行WSSV检测,显示携带量分别为17.79±3.71 copies/ng DNA和19.06±4.31 copies/ng DNA。设计三种饵料组合,即高锰酸钾消毒的菲律宾蛤仔足肌+沙蚕+配饵（消毒组）,菲律宾蛤仔足肌+沙蚕+配饵（对照组）及配饵（配饵组）,分别投喂对虾60天。结果表明:越冬期间投喂消毒组、对照组和配饵组这3种饵料组合的对虾体重量分别降低了1.48±0.86g、0.39±0.63g和1.71±0.91g,投喂三种饵料的对虾存活率分别为52.53%、79.75%和46.01%。投喂消毒组、对照组与配饵组饵料的对虾越冬前WSSV携带量分别为7.63±6.24 copies/ng DNA、10.58±7.34 copies/ng DNA和11.97±9.32 copies/ng DNA,越冬后分别为11.69±9.18 copies/ng DNA、9.31±6.95 copies/ng DNA和16.87±14.47 copies/ng DNA,越冬前后变化差异不显著（P>0.05）。亲虾在产卵量、受精率和孵化率三个指标上的排序均为对照组>消毒组>配饵组,且均呈显著差异（P<0.05）。研究结果表明,菲律宾蛤仔和沙蚕不同程度的携带WSSV,利用对照组饵料投喂亲虾,可获得较高的产卵量、受精率和孵化率,但投喂上述三组饵料,越冬前后对虾体内的WSSV携带量变化差异不显著。3、不同饵料组合对中国对虾幼体WSSV携带量及存活率的影响采用单胞藻（SA）、配合饵料（AF）、轮虫（BP）和卤虫幼体（BS）4种饵料,设计了SA+AF、SA+AF+BP、SA+AF+BS和SA+AF+BP+BS等4种饵料组合并用于对虾苗种培育,其中投喂SA+AF饵料组合的受精卵分设“碘伏”消毒及未消毒组,投喂其它饵料组合的受精卵均为“碘伏”消毒组。观察和分析各期幼体成活率,结果表明,N-Z期间,受精卵消毒与否及投喂不同饵料组合对对虾的成活率差异均不显著（P>0.05）;Z-P期间,投喂BP的成活率明显高于未投喂BP组（P<0.01）;饵料组合SA+AF+BS在M-P期间幼体成活率与其它饵料组差异极显著（P<0.01）。对P₁₀幼体体重测量结果表明,投喂SA+AF+BP的虾苗平均体重小于投喂SA+AF+BP+BS的幼体（P<0.01）。P₁₀仔虾WSSV携带量检测结果表明, SA+AF消毒组仔虾WSSV携带量为10.52±3.3 copies/ng DNA,显著低于其它各组饵料培育的仔虾WSSV携带量（P<0.05）。在P₁₁-P60培育期间,分别投喂菲律宾蛤仔足肌（CF）+配饵（AF）、⁶⁰Coγ辐照菲律宾蛤仔足肌（RCF）+配饵（AF）、高锰酸钾消毒菲律宾蛤仔足肌（DCF）+配饵（AF）和配饵（AF）4种饵料组合。结果表明, CF+AF组对虾体重和体长增长最大,但与AF组差异不显著,而CF+AF组和AF组与其余两组差异显著（P<0.05）;CF+AF组存活率最高,但与其余3组差异不显著（P>0.05）。WSSV人工感染实验结果表明,四种饵料投喂的对虾累积死亡率都在90%以上（P>0.05）,显示⁶⁰Coγ辐照（RCF）和高锰酸钾消毒后的菲律宾蛤仔足肌（DCF）投喂P₁₁-P₆₀对虾幼体,对感染WSSV后的对虾存活率无显著影响。4、中国对虾的WSSV携带量与生长及抗WSSV育种值的相关性分析对130个中国对虾家系进行生长及抗WSSV能力测试。根据统计软件分析结果,选择出生长育种值最大的5个家系和最小的5个家系,以及抗WSSV育种值最大的5个家系和最小的5个家系,分别检测上述20个家系的亲虾以及养殖50 d和170 d时对虾的WSSV携带量。结果表明,亲虾、50 d对虾及170 d对虾的WSSV携带量分别为0.1908 copies /ng DNA、0.2866 copies /ng DNA和0.2329 copies /ng DNA,三者之间差异均不显著（P>0.05）。亲虾、50 d对虾和170 d对虾的WSSV携带量与对虾的生长育种值相关系数分别为0.021、0.363和0.185（P>0.05）,亲虾、50 d对虾和170 d对虾的WSSV携带量与抗WSSV育种值相关性分别为0.033、0.048和0.019（P>0.05）,显示中国对虾的生长育种值和抗WSSV育种值与对虾体内的WSSV携带量均无显著的相关性。5、结论及研究的意义三种PCR方法检测WSSV的灵敏度由高到低依次为:TaqMan实时定量PCR、巢式PCR、一步法PCR;中国对虾WSSV携带量与其繁殖和育种值之间均无显著相关性,与其存活率之间存在一定的相关性。本研究为中国对虾WSSV疫情的预防及治理提供了理论及实际基础。更多还原

【Abstract】 The epidemic pathogenic of Fenneropenaeus chinensis is main caused by White spot syndrome virus(WSSV), no effectively technical measures could control the WSSV epidemic so far. For the purpose of prevention and decrease of WSSV loss to F. chinensis, first of all we compared the sensitivity of three PCR methods on detecting the white spot syndrome virus (WSSV); and then we focused on analysing the correlation of WSSV load and reproduction、survival and breeding value in F. chinensis. The results are as following:1、Sensitivity comparison of three different kinds of PCR-based methods in white spot syndrome virus (WSSV) testIn this part, we compared the sensitivity of several PCR methods, including TaqMan real-time PCR, Office International Des Epizooties (OIE) and Genetic resource and Our Lab-designed nested PCR (GB) and two corresponding one-step PCR methods, with standard samples of serial known WSSV load of F. chinensis. As a result, TaqMan real-time PCR could detect with certainty 10 WSSV copies per reaction while 10~4, 10~3 copies were needed for OIE and GB nested PCR; 5×10~4, 2.5×10~4 copies were needed for the outer primer and inner primer amplification separately of OIE nested PCR; and 10~4, 5×10~3 copies were needed for that of the GB nested PCR. 44 F. chinensis samples which we did not know the WSSV load were tested with these PCR methods to validate the sensitivity, the positive ratio of TaqMan real-time PCR was 84.09 %, and that of the OIE and the GB nested PCR were 18.18% and 27.27%, respectively; the positive ratio of the outer primer and inner primer amplification separately of OIE nested PCR both were 15.91%; and that of the GB nested PCR were 18.18% and 20.45%. According to the results, the sensitivity of PCR detected the WSSV from high to low are: TaqMan real-time PCR, nested PCR, one-step PCR.2、Effect of different diets on the WSSV loads and reproduction of F. chinensis over winterBased on the detecting result of WSSV loads of two kinds of prawn overwinter diets , Ruditapes philippinarum and clamworm, which were 17.79±3.71 copies/ng DNA and 19.06±4.31 copies/ng DNA, respectively, three kinds of diet combinations were designed as 1) disinfected Ruditapes philippinarum musle + disinfected clamworm + artificial diets (disinfected group); 2) R. philippinarum musle + clamworm + artificial diets (control group) ; 3) artificial diets (artificial group). After 60 days it was showed that the loss of weight of prawn in three groups was 1.48±0.86g、0.39±0.63g and 1.71±0.91g; and the survival rate of each group was 52.53%、79.75% and 46.01%. The WSSV loads of prawn before and after overwinter were 7.63±6.24 copies/ng DNA、10.58±7.34 copies/ng DNA、11.97±9.32 copies/ng DNA and 11.69±9.18 copies/ng DNA、9.31±6.95 copies/ng DNA、16.87±14.47 copies/ng DNA, respectively, showing no significant difference (P>0.05). The fecundity、the rates of fertilization and hatching of the brookstock from high to low were group of feeding control group、disinfected group and artificial group, showing significant difference (P<0.05). It was indicated that the R. philippinarum and clamworm can carry small amount of WSSV, Besides this, the brookstock can gain high fecundity、high rates of fertilization and hatching when feeding on control group diet combination over winter, the WSSV load of prawn feeding on three diet combinations is showing no significant difference overwinter.3、Effect by feeding different diets on the WSSV load and survival rate of F. chinensis.Based on single-cell algae (SA), artificial feed (AF), brachionus plicatilis (BP) and brine shrimp larva (BS), four groups of diet combination were designed as SA + AF, SA + AF + BP, SA + AF + BS, SA + AF + BP + BS , and used to the cultivation of F. chinensis larval, the oosperm which feeding on the group SA+AF divided into disinfected and undisinfected group, other groups’oosperm all were disinfected group. The survival rate of each stage was compared in different groups. It was showed that there was no significant difference from each other in the survival rate of N-Z by diet different diet combination (P>0.05). The survival rate of Z-P fed BP was higher than others (P<0.01) and the survival rate of M-P fed SA + AF + BS was significant difference (P<0.01) from others. The body weight of 10-day post larva by diet SA + AF + BP was less than that by diet SA + AF + BP + BS (P<0.01). The result of detecting WSSV load of 10-day post larva were disinfection Group SA + AF WSSV larva carrying the lowest load of 10.52±3.3 WSSV copies / ng DNA, which lower than other diet groups (P<0.05 ).During the stage of P11-P60, R. philippinarum foot muscle (CF) + artificial feed (AF), 60Coγradiated R. philippinarum foot muscle (RCF) + artificial feed (AF), potassium-permanganate-disinfected R. philippinarum foot muscle (DCF) + artificial feed (AF) and artificial feed (AF), which four diet groups were feeding to prawn. Shrimp of group CF + AF gained the largest increase of body weight and body length and showed no significantly difference (P>0.05) from group AF, but showed significantly difference (P<0.05) from the other two groups. The survival rate of group CF + AF was highest, (P>0.05) The result of WSSV artificial infection experiment showed that all groups of shrimp accumulated mortality were higher than 90% (P>0.05). It was concluded that shrimp larval feeding on 60Coγradiated R. philippinarum foot muscle (RCF) and potassium-permanganate-disinfected R. philippinarum foot muscle (DCF) could not caused significant effect of survival rate.4、The relationship between viral loads and growth & WSSV resistanceAfter testing the growth and WSSV resistance of 130 F. chinnensis families, According to the results of statistics software analysis, we choosed the five families each of largest and smallest growth breeding value, five families each of largest and smallest WSSV-resistance breeding value.We detecting the WSSV load of these broodstock, 50 d shrimp and 170 d shrimp of 20 families. The mean WSSV load of broodstock, 50 d shrimp and 170 d shrimp of 20 families were 0.1908, 0.28662 and 0.2329 copies / ng DNA, respectively, which showed no significant difference (P>0.05). The correlation coefficient between WSSV-resistance breeding value and WSSV load of broodstock, 50 d shrimp and 170 d shrimp were 0.021, 0.463 and 0.185, respectively; and that between growth breeding value and WSSV load of broodstock, 50 d shrimp and 170 d shrimp were 0.033, 0.048 and 0.019, respectively. The results shows that the relationship between growth breeding value, WSSV-resistance breeding value and the WSSV load of shrimp were not significant (P>0.05).5.Conclusion and research implicationsThe sensitivity of PCR detected the WSSV from high to low are: TaqMan real-time PCR, nested PCR, one-step PCR; The correlation of WSSV load and reproduction、breeding value in F. chinensis all showing no significant difference, The correlation of WSSV load and suivival is showing certain significant difference. This research provided the theoretical and practical basis in the prevention and management WSSV epidemic of F. chinnensis.更多还原