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

首先，我们开发了一种从咽喉冲洗样本中提取的简便易行的方法，这是一种适合重复采集的样本，并且侵袭性较小，用于检测疱疹病毒群病毒。用商品化的DNA提取试剂盒提取50m1以下的血液，与超速离心法和人工乙醇沉淀法相比，在不降低DNA得率的情况下，大大缩短了DNA提取时间。随后，我们利用针对EBV-W、BNRF-1和bc1-2基因的引物和探针，建立了实时荧光定量检测咽拭子标本中EB病毒DNA拷贝数的方法。为了获得高精度的检测结果，我们用聚合酶链式反应和TA克隆的方法制备了阳性对照的引物(bc1-2、EB病毒W、EB病毒BNRF-1)，建立了以实时荧光定量检测为标准的高效定量检测体系。收集了33例健康…患者的582份长期咽喉冲洗标本多行扁桃体切除术47例。由于提供了Real-Time PCR，我们根据年龄、临床诊断等临床数据进行分析。分析结果表明，所有临床标本中的洗喉液、血液中的含污量没有显著差异。作为临床应用，我们对530例疑似淋球菌或沙眼衣原体性传播感染的患者进行了洗喉液与咽拭子检测性能的比较。感染。对每例患者的咽拭子和咽拭子同时进行检测，并用双核酸扩增法进行检测。在每次扩增和每种微生物的检测结果中，咽拭子法优于咽拭子法。这些结果表明，洗喉可以作为口咽部微生物检测的样本。此外，通过漱口、前后进食的方式采集的DNA数量不均匀，有必要重新考虑仔细设置洗喉，以应对未来的检查问题。较少

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