Molecular studies on herpes group virus detection with throat wash to apply in a clinical setting

首先，我们开发了一种简单易行的从洗喉样本中提取的方法，该方法是一种适合反复提取的样本，具有较小的侵袭性，用于检测疱疹群病毒。在使用小于50m1血液的商业DNA提取试剂盒时，我们能够大大缩短DNA提取时间，而不降低DNA产量，与使用超离心和人工乙醇沉淀的细胞分离DNA提取相比。随后，我们利用EBV- w、BNRF-1和bcl-2基因的引物和探针，制备了用实时荧光定量PCR （Taqman）技术对洗喉样本进行EBV DNA拷贝数定量检测的系统。为了获得较高的测量精度，我们对引物（bcl-2、EBV W、EBV BNRF-1）进行PCR和TA克隆建立了阳性对照，并用质粒稀释溶液作为Real-Time PCR标准物建立了高定量的测量体系。我们从33名健康受试者和47名扁桃体切除术患者中收集了582份随时间推移的洗喉样本。由于提供了Real-Time PCR，我们根据年龄、临床诊断等临床数据对其进行分析。分析结果显示，在所有临床样本中，所有个体的咽洗液和血液中，多巴胺的数量没有显著差异。作为临床应用，我们对530例疑似淋病奈瑟菌或沙眼衣原体性传播感染的患者进行了咽洗液与咽拭子的对比检测。感染。每个病例的洗喉和咽拭子同时检测，用两次核酸扩增法检测。洗喉法在各扩增物和各微生物的检测次数上均优于咽拭子法。这些结果表明，洗喉可能是有用的样品，从口咽作为物的微生物检测。此外，通过漱口方式收集的DNA数量不均匀，前后喂食，并且重新考虑仔细设置以进行喉咙清洗是将来检查问题所必需的。少

As first, we developed a simple and easy protocol to extract from throat washing samples which is an appropriate sample to take repeatedly with less aggressions for detection of herpes group virus. In using a commercial DNA extraction kit for blood less than 50m1, we were able to largely shorten DNA extraction time without decreasing a DNA yield in comparison to cell separation DNA extract with super centrifuging and ethanol precipitation by manual labor. Subsequently we prepared systems to detect the DNA copy numbers of EBV quantitatively with Real-Time PCR (Taqman) for the throat washing samples by using primers and probes for EBV-W, BNRF-1,and bcl-2 gene. For measurement with high precision, we producted of positive control ith PCR and TA cloning of the primers (bcl-2, EBV W, EBV BNRF-1) and established the high fixed-quantity measurement system using dilute solutions of the plasmids for standard of Real-Time PCR.We collected 582 throat washing samples taked over time from 33 health … More y subject and 47 cases performed tonsillectomy. As a result of having been provided with Real-Time PCR, we analyzed them according to clinical data such as age, clinical diagnosis. The results of analysis showed no significant differences in show no significant differences in dopy number among all clinical samples which were throat washings, blood in all individuals.As the clinical application using throat samples washings, we weigh detection performance of throat washings against throat swabs in 530 cases suspected a sexually transmitted infection with Neisseria Gonorrhoeae or Chlamydia trachomatis. infection. The throat washing and throat swab of each case teked simultaneously and examined with two nucleic acid amplification method. The throat washing was superior to the throat swab in number of detection performance with each amplification and each microorganism. These results suggest that throat washing may be useful as a sample for the microbe detection from oropharynx as things.In addition, unevenness was shown to quantity of DNA collected in way of a gargle, feeding front and back, and reconsideration of careful setting to take a throat washing was necessary with a future examination problem. Less