2005 Fiscal Year Final Research Report Summary
Molecular studies on herpes group virus detection with throat wash to apply in a clinical setting
| Project/Area Number |
14571640
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | Tokyo Women's Medical University |
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Principal Investigator |
YODA Keiko Tokyo Women's Medical University, School of Medicine, Assistant Proffesor, 医学部, 講師 (70240364)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Nobuaki Tokyo Women's Medical University, School of Medicine, Physician Assistant, 医学部, 助手 (50297553)
UCHIMURA Kanako Tokyo Women's Medical University, School of Medicine, Physician Assistant, 医学部, 助手 (30343552)
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| Project Period (FY) |
2002 – 2005
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| Keywords | Oropharynx / Throat wash / Real time PCR / EBV / Neisseria Gonorrhoeae / Chlamydia trachomatis / HHV-7 / in situ hybridization |
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| Research Abstract |
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
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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
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