Joss A W, Ho-Yen D O
Scottish Toxoplasma Reference Laboratory, Raigmore Hospital NHS Trust, Inverness.
J Med Microbiol. 1997 Jan;46(1):92-6. doi: 10.1099/00222615-46-1-92.
The effects of sample storage, target preparation and annealing temperature on a nested polymerase chain reaction (PCR) test for Toxoplasma gondii DNA were investigated with experimentally seeded amniotic fluids to simulate congenital infection. Aliquots of 17 amniotic fluid samples remaining after investigation for conditions other than toxoplasmosis and seeded to contain small numbers of T. gondii tachyzoites, were tested after storage for up to 2 weeks. There was no deterioration in test sensitivity on samples stored for 1-2 weeks at room temperature; thus, samples sent by post to reference laboratories are acceptable for examination. There was no significant difference in the results from two target preparation methods or two different annealing temperatures. One-to-ten parasites were detectable in 13 of 17 test samples. The significant feature in the remaining four samples was the use of washed stored parasites to seed the amniotic fluids rather than any feature of the amniotic fluids used or the test. False positive results were found in up to 15% of unseeded amniotic fluid samples, which contrasts with only 1.9% for the same PCR test in other routine or negative control samples tested as part of the laboratory's reference diagnostic work. The difference illustrates the increased risk of cross-contamination in PCR when the majority of specimens tested are positive. The results suggest that PCR techniques are likely to be sensitive and effective tools for the routine diagnosis of congenital toxoplasma infection.
为模拟先天性感染，使用实验接种的羊水研究了样本储存、靶标制备和退火温度对弓形虫DNA巢式聚合酶链反应（PCR）检测的影响。对17份羊水样本进行了除弓形虫病以外其他情况的检测，剩余样本接种少量弓形虫速殖子，储存长达2周后进行检测。在室温下储存1至2周的样本，检测灵敏度没有下降；因此，通过邮寄送往参考实验室的样本可接受检测。两种靶标制备方法或两种不同退火温度的检测结果没有显著差异。17个检测样本中有13个可检测到1至10个寄生虫。其余4个样本的显著特点是使用洗涤过并储存的寄生虫接种羊水，而非所用羊水或检测的任何特点。在高达15%的未接种羊水样本中发现假阳性结果，相比之下，在作为实验室参考诊断工作一部分检测的其他常规或阴性对照样本中，相同PCR检测的假阳性率仅为1.9%。这种差异表明，当检测的大多数标本为阳性时，PCR检测中交叉污染的风险增加。结果表明，PCR技术可能是先天性弓形虫感染常规诊断的灵敏且有效的工具。
