对虾6种病毒多重PCR检测方法的建立

Multiplex PCR assay for simultaneous detection of six viruses in shrimp

本研究针对养殖对虾6种病毒,包括白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、肝胰腺细小病毒(HPV)、桃拉综合征病毒(TSV)、对虾杆状病毒(BP)和传染性肌肉坏死病毒(IMNV),选择各自的基因分别设计特异性引物和探针,首先进行了单一病毒的PCR验证,在此基础上建立了同时特异性检测6种对虾病毒的多重PCR检测体系。对反应条件进行优化并进行特异性和灵敏度的验证。50μl反应体系,Mg2+的最佳浓度为5mmol/L,ExTaq酶最佳用量为3.75U,反应程序中最佳退火温度为55.5℃。6种病毒之间以及与对虾基因组都存在很好的特异性。最终经试验验证,该系统的检测灵敏度对WSSV可达104拷贝,IHHNV可达102拷贝,HPV可达104拷贝,TSV可达103拷贝,BP可达105拷贝,IMNV可达105拷贝。虽然该多重PCR方法灵敏度不如单一的PCR检测高,但是通过实际样品检测验证了该方法省时、消耗较少,又不失准确性,在实际应用中具有可靠性和应用价值。

In this study, we developed a multiplex reverse transcription-polymerase chain reaction for simultaneous detection of six major shrimp viruses including white spot syndrome virus （WSSV）, infectious hypodermal and hematopoietic necrosis virus （IHHNV）, hepatopan creatic parvo virus （HPV）, Taura syndrome virus （TSV）, Baculowirus penaei （BP） and infec tious myonecrosis virus （IMNV）. The reaction condition was optimized, and the specificity and sensitivity of Ibis method were tested. In 50 μl reaction system, the optimum concentration of Mg2＋ was 5mmol/L, the optimum dosage of gxTaq enzyme was 3.75U, and the optimal annea- ling temperature was 55.50℃. There was good specificity between the six viruses and shrimp ge nome. The detection limits were 104copies for the detection of WSSV, 102copies for IHHNV, 104copies for HPV, 103copies for TSV, 105copies for BP, and 105copies for IMNV. Although the multiplex PCR detection is not as sensitive as a single PCR, the actual sample testing valida- ted that the method is time-saving and less reagent consuming.