Smith J J, Gunasekera T S, Barardi C R M, Veal D, Vesey G
Montana Microbiological Services, IIc., Bozeman, MT, USA.
J Appl Microbiol. 2004;96(2):409-17. doi: 10.1046/j.1365-2672.2004.02150.x.
Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum.
FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h.
Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity.
Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.
荧光原位杂交技术（FISH）已被用于微小隐孢子虫卵囊的种特异性检测和活力测定。基于FISH的活力测定依赖于活力丧失后rRNA的降解。我们研究了核糖核酸酶和核糖核酸酶抑制剂对微小隐孢子虫FISH的影响。
使用浓度为1 pM μl-1的5'-德克萨斯红标记的DNA寡核苷酸探针进行FISH。完整的和经热通透处理的卵囊用1-100 μg ml-1的核糖核酸酶处理。如果在通透处理前将外源性核糖核酸酶中和，完整卵囊的FISH似乎不受影响。室温下储存在磷酸盐缓冲盐水中的热灭活卵囊，其FISH荧光在55小时后衰减一半，但在6天后仍可检测到。添加钒核糖核苷复合物（VRC）可将热通透处理卵囊的rRNA半衰期延长至155小时。
延长的rRNA半衰期可能导致使用FISH高估活力。建议在FISH之前进行核糖核酸酶预处理，以破坏最近灭活卵囊中残留的rRNA。在FISH通透处理步骤之前加入1-10 mM l-1的VRC应能中和核糖核酸酶活性。
消除非存活微小隐孢子虫的FISH荧光是可取的。建议使用核糖核酸酶和VRC来减少假阳性“存活”卵囊的数量。
