Rajendran K V, Cowley Jeff A, McCulloch Russell J, Walker Peter J
CSIRO Food Futures Flagship, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia 4067, Australia.
J Virol Methods. 2006 Nov;137(2):265-71. doi: 10.1016/j.jviromet.2006.06.026. Epub 2006 Aug 7.
A highly sensitive and specific TaqMan real-time quantitative RT-PCR (qRT-PCR) was developed to detect and quantify Mourilyan virus (MoV), a newly described bunya-like virus of penaeid shrimp. The PCR primers and TaqMan probe targeted a 67-nucleotide (nt) sequence in the MoV M RNA segment. Using dilution series of a 849 nt RNA transcribed in vitro from cDNA clone pMoV4.1, the assay could detect down to a single MoV RNA equivalent, reliably detected 10 RNA copies and had a log linear range up to 1 x 10(9) RNA copies. In experimentally infected Penaeus japonicus shrimp, the test was used to quantify increases in MoV loads over time in hemocytes, lymphoid organ and gills. Sequential increases in MoV RNA copy numbers occurred in lymphoid organ and gill tissues collected at 6, 24 and 48 h post-infection. However, RNA copy numbers decreased slightly in hemocytes sampled at 48 h compared to 24 h. The qRT-PCR data correlated well with amplicon yields generated using a conventional RT-nested PCR targeting the same MoV RNA segment. Moreover, histology and in situ hybridisation using shrimp cephalothorax sections identified increases in lymphoid organ spheroid numbers and confirmed that increases in MoV RNA detected in lymphoid organ tissue were due to expansion in the numbers of infected cells. The qRT-PCR assay should find use in high-throughput screening applications to detect MoV in broodstock and postlarvae used for culture or breeding purposes and for tracking changes in infection levels during shrimp grow-out.
开发了一种高灵敏度和特异性的TaqMan实时定量逆转录聚合酶链反应（qRT-PCR），用于检测和定量穆里扬病毒（MoV），这是一种新描述的对虾类布尼亚样病毒。PCR引物和TaqMan探针靶向MoV M RNA片段中的一段67个核苷酸（nt）的序列。使用从cDNA克隆pMoV4.1体外转录的849 nt RNA的稀释系列，该检测方法可检测到低至单个MoV RNA等效物，可靠地检测到10个RNA拷贝，并且对数线性范围高达1×10⁹个RNA拷贝。在实验感染的日本对虾中，该检测方法用于定量血细胞、淋巴器官和鳃中MoV载量随时间的增加。在感染后6、24和48小时收集的淋巴器官和鳃组织中，MoV RNA拷贝数依次增加。然而，与24小时相比，48小时采集的血细胞中RNA拷贝数略有下降。qRT-PCR数据与使用针对相同MoV RNA片段的常规逆转录巢式PCR产生的扩增子产量相关性良好。此外，使用对虾头胸部切片的组织学和原位杂交确定了淋巴器官球体数量的增加，并证实淋巴器官组织中检测到的MoV RNA增加是由于受感染细胞数量的增加。qRT-PCR检测方法应可用于高通量筛选应用，以检测用于养殖或繁殖目的的亲虾和后期幼体中的MoV，并跟踪对虾养成期间感染水平的变化。
