实时荧光定量PCR检测克氏原螯虾体内白斑症病毒(WSSV)的动态变化

Analysis on Dynamic Changes of WSSV Amount in Procambarus clarkii through Quantitative Real-time PCR

本文建立了白斑症病毒（WSSV）实时荧光定量PCR标准曲线，结果显示其线性关系良好，利用建立的WSSV实时荧光定量PCR技术检测感染了WSSV后克氏原螯虾（Procambarus clarkii）组织内病毒数量的时序动态变化。注射WSSV粗提液感染克氏原螯虾，在感染后0h、3h、6h、12h、24h、36h、48h、60h和72h分别取螯虾鳃、血淋巴、心脏、附肢、造血组织和肌肉，提取组织DNA，实时荧光定量PCR测定WSSV浓度，发现感染后3h在附肢、鳃、血淋巴和造血组织中检测到病毒，12h时在各组织中皆检测到病毒，但浓度较低；24~48h内WSSV浓度呈指数增长，此时螯虾出现少量死亡；60~72h内WSSV数量缓慢增长达到平台期，螯虾累计死亡率快速升高。不同组织中的WSSV浓度有较大差异，附肢中浓度最高，血淋巴、鳃、心脏和造血组织稍低，肌肉中WSSV浓度最低；附肢中WSSV动态变化代表了WSSV增殖变化规律，是很好的病毒检测与病程进展评估材料。本文结果可为WSSV的早期诊断提供技术支持，为阐明WSSV感染增殖规律提供资料。

A real-time quantitative PCR was developed and the absolute copy number of WSSV VP28 gene was quan-tified based on a standard curve in Procambarus clarkii infected with WSSV. Following intramuscular injection with WSSV,the gill,hemolymph,heart,muscle,pleopods and hemopoietic tissue of Procambarus clarkii were sampled at 0,3,6,12,24,36,48,60 and 72 h post infection（hpi）,DNA was extracted from the samples and the viral load was analyzed. It was found that WSSV was detected in pleopod,gill hemolymph and hemopoietic tissue at 3 hpi, and in all sampled tissues at 12hpi with low absolute copy number of WSSV;then the WSSV copies increased logarith-mically from 24 to 48hpi and a low mortality rate was observed. Thereafter,the WSSV propagation went into the pla-teau phase and massive mortality appeared from 60 to 72hpi,showing a positive correlation between the viral load and cumulative mortality of Procambarus clarkii. Obvious difference in WSSV copy numbers were found in tested tissues, and WSSV copies in pleopod were the highest,followed by hemolymph,gill,heart and hemopoietic tissue,and the lowest in muscle. The dynamic changes of the amount of WSSV in pleopods could well represent all sampled tissues of Procambarus clarkii,so pleopods could be a reliable tissue for WSSV detection. This study might provide technical support for early diagnosis of WSSV infection and important basic data for better understanding of WSSV proliferation rules in shrimp.