Milks Maynard L, Sokolova Yuliya Y, Isakova Irina A, Fuxa James R, Mitchell Forrest, Snowden Karen F, Vinson S Bradleigh
Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
J Eukaryot Microbiol. 2004 Mar-Apr;51(2):187-91. doi: 10.1111/j.1550-7408.2004.tb00544.x.
The main goal of this study was to compare the effectiveness of three staining techniques (calcofluor white M2R, Giemsa and modified trichrome), and the polymerase chain reaction (PCR) in detecting the microsporidium Thelohania solenopsae in red imported fire ants (Solenopsis invicta). The effect of the number of ants in a sample on the sensitivity of the staining techniques and the PCR, and the effect of three DNA extraction protocols on the sensitivity of PCR were also examined. In the first protocol, the ants were macerated and the crude homogenate was used immediately in the PCR. In the second protocol, the homogenate was placed on a special membrane (FTA card) that traps DNA, which is subsequently used in the PCR. In the third protocol, the DNA was purified from the homogenate by traditional phenol-chloroform extraction. Except for PCR using FTA cards, the sensitivity (number of samples positive for T. solenopsae) of all detection techniques increased with the number of ants in the sample. Overall, Giemsa was the least sensitive of all detection techniques. Calcofluor was more sensitive than modified trichrome with ants from one site and was equally as sensitive as PCR with crude DNA or a FTA card with ants from both sites. Trichrome staining was equally as sensitive as PCR with a FTA card at both sites, but it was less sensitive than PCR with crude DNA at one site. PCR on FTA cards was less sensitive than PCR with crude DNA for ants from one site but not the other. There was no difference whether crude or phenol-chloroform purified DNA was used as template. In summary, the results of this study show that PCR based on a crude DNA solution is equal to or more sensitive in detecting T. solenopsae than the other detection techniques investigated, and that it can be used as a reliable diagnostic tool for screening field samples of S. invicta for T. solenopsae. Nevertheless, ant smear stained with calcofluor or modified trichrome should be used to buttress findings from PCR.
本研究的主要目标是比较三种染色技术（荧光增白剂M2R、吉姆萨染色和改良三色染色）以及聚合酶链反应（PCR）检测红火蚁（Solenopsis invicta）体内微孢子虫Thelohania solenopsae的效果。同时还研究了样本中蚂蚁数量对染色技术和PCR灵敏度的影响，以及三种DNA提取方法对PCR灵敏度的影响。在第一种方法中，将蚂蚁研磨成匀浆，所得粗匀浆直接用于PCR。在第二种方法中，将匀浆置于一种能捕获DNA的特殊膜（FTA卡）上，随后将捕获的DNA用于PCR。在第三种方法中，通过传统的酚-氯仿提取法从匀浆中纯化DNA。除了使用FTA卡进行的PCR外，所有检测技术的灵敏度（感染Thelohania solenopsae的样本数量）均随样本中蚂蚁数量的增加而提高。总体而言，吉姆萨染色是所有检测技术中灵敏度最低的。荧光增白剂染色在检测来自一个地点的蚂蚁时比改良三色染色更灵敏，在检测来自两个地点的蚂蚁时，其灵敏度与使用粗DNA或FTA卡进行PCR的灵敏度相当。三色染色在两个地点的灵敏度与使用FTA卡进行PCR的灵敏度相当，但在一个地点比使用粗DNA进行PCR的灵敏度低。对于来自一个地点而非另一个地点的蚂蚁，在FTA卡上进行PCR的灵敏度低于使用粗DNA进行PCR的灵敏度。使用粗DNA或酚-氯仿纯化的DNA作为模板并无差异。总之，本研究结果表明，基于粗DNA溶液的PCR在检测Thelohania solenopsae方面等同于或比其他所研究的检测技术更灵敏，并且它可作为一种可靠的诊断工具，用于筛查红火蚁野外样本中的Thelohania solenopsae。尽管如此，用荧光增白剂或改良三色染色的蚂蚁涂片应用于辅助PCR检测结果。
