Hasson K W, Hasson J, Aubert H, Redman R M, Lightner D V
Department of Veterinary Science, University of Arizona, Tucson 85721, USA.
J Virol Methods. 1997 Jul;66(2):227-36. doi: 10.1016/s0166-0934(97)00066-9.
In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH approximately 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.
利用桃拉综合征病毒（TSV）特异性cDNA探针，对虾组织切片进行原位杂交分析，是目前可用于检测这种对虾病毒性疾病的最灵敏诊断技术。然而，对于用戴维森AFA（乙酸、甲醛、酒精）固定剂保存的凡纳滨对虾样本，经常会得到假阴性的基因组探针结果，而这些样本在常规组织学检查中显示出典型的TSV病变。这个问题与虾样本在高度酸性（pH约3.5 - 4）的戴维森固定剂中长时间保存有关。推测TSV基因组RNA的降解是由于固定剂诱导的酸水解和/或嗜酸性内源性核糖核酸酶活性。对等量感染TSV的凡纳滨对虾幼体进行常规苏木精和伊红（H和E）组织学检查以及原位杂交分析，这些幼体分别用戴维森固定剂或作者开发的一种新的近中性（pH约6.0 - 7.0）、对RNA友好的固定剂（R - F）保存四个不同时间段（2、6、10和14天）。原位杂交试验在有和没有核糖核酸酶预防措施的情况下进行，所有测试样本通过常规组织学检查都含有中度至重度TSV病变。用戴维森固定剂保存的样本在固定2天后产生微弱的TSV探针信号，但在固定剂中保存超过6天的样本中不与探针反应。相比之下，在用新的R - F固定剂保存的所有时间处理样本中，基因探针都能检测到TSV。在没有无核糖核酸酶条件下分析相同样本时，获得了等效的原位杂交结果。这些发现表明，当样本在酸性固定剂（如戴维森固定剂）中保存超过2天时，TSV RNA会降解，并且通过用R - F固定剂保存虾样本可以防止这个问题。证明了这种新固定剂的有效性，结果表明进行TSV原位杂交分析不需要无核糖核酸酶条件。
