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

中文关键词: WSSV  IHHNV  HPV  TSV  BP  IMNV  多重PCR

英文关键词: WSSV  IHHNV  HPV  TSV  BP  IMNV  Multiplex PCR

中文摘要:

      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六种对虾病毒的多重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。50 µl反应体系，Mg²⁺的最佳浓度为5 mmol/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。虽然灵敏度不如单一的PCR检测的灵敏度高，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，在实际应用中的可靠性和应用价值。

英文摘要:

      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), hepatopancreatic parvo virus (HPV), Taura syndrome virus (TSV), Baculovirus penaei (BP) and infectious myonecrosis virus (IMNV). The reaction condition was optimized, and the specificity and sensitivity was tested for this method. In 50 µl reaction system, the optimum concentration of Mg²⁺ was 5mmol/L, optimum dosage of ExTaq enzyme was 3.75U, reaction procedure in the optimal annealing temperature was 55.5℃. Between six kinds of virus and shrimp genome exist good specificity. The detection limits were 104copies for the detection of WSSV, 10² copies for IHHNV, 10⁴ copies for HPV, 10 ³ copies for TSV, 10⁵ copies for BP, and 10⁵ copies for IMNV. Although the sensitivity is not as good as a single PCR,the actual sample testing validated that the method time-saving and less reagent consuming.