Sakuna Kitikarn, Elliman Jennifer, Owens Leigh
College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, 4811, Australia; Faculty of Veterinary Science, Rajamangala University of Technology Srivijaya, 80240, Thailand.
College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, 4811, Australia.
J Virol Methods. 2018 Jan;251:139-144. doi: 10.1016/j.jviromet.2017.10.013. Epub 2017 Oct 17.
Chequa iflavirus (+ve sense ssRNA virus) infects redclaw crayfish (Cherax quadricarinatus) and it may cause mortality reaching 20-40% after about three weeks following stress. The sequence of the RNA-dependent RNA polymerase at nucleotide position 8383-9873 was used for developing and comparing PCR-based detection protocols. The reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) was specific against nine Picornavirales and crustacean viruses and its' measurement of uncertainty (0.07-1.37) was similar to PCRs for other crustacean viruses. In vitro, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) read at 60min had poor repeatability for a linearized plasmid with an iflavirus insert when compared with RT-PCR visualised on an electrophoretic gel and RT-qPCR; both sensitive to 10 copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88×10 for RT-PCR and 4.60×10 for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14×10. For RT-qPCR, the amplification plots, melt curves and the C values indicated that the C above 34.0 is a potential negative result but examination of the melt curve is necessary for an accurate interpretation. A suggested program of testing for crayfish farmers would consist of non-destructive bleeding, labelling of crayfish and screening with RT-qPCR. Only those crayfish nominally negative (below detectable limits) would be used for broodstock or selective breeding.
切夸伊黄病毒（正链单链RNA病毒）感染红螯螯虾（四脊光壳螯虾），在应激后约三周可能导致死亡率达到20%-40%。利用核苷酸位置8383-9873处的RNA依赖性RNA聚合酶序列来开发和比较基于PCR的检测方案。逆转录定量聚合酶链反应（RT-qPCR）对九种小RNA病毒目病毒和甲壳类病毒具有特异性，其测量不确定度（0.07-1.37）与其他甲壳类病毒的PCR相似。在体外，与在电泳凝胶上可视化的RT-PCR和RT-qPCR相比，逆转录环介导等温扩增（RT-LAMP）在60分钟时对带有伊黄病毒插入片段的线性化质粒的重复性较差；两者对10个拷贝均敏感。在有限的临床小龙虾血淋巴比较样本中，RT-PCR的最低非零拷贝数为2.88×10，RT-qPCR为4.60×10。在仅通过RT-qPCR检测的另外68份临床小龙虾血淋巴样本中，拷贝数范围为0至1.14×10。对于RT-qPCR，扩增曲线、熔解曲线和C值表明，C值高于34.0可能为阴性结果，但准确解读需要检查熔解曲线。建议小龙虾养殖户的检测方案包括无损采血、小龙虾标记以及用RT-qPCR进行筛查。只有那些名义上为阴性（低于检测限）的小龙虾才会用于亲虾或选择性育种。
