イベント・セミナー詳細

専門家

10月水曜会(第670回)

情報公開日:2011年10月20日 (木曜日)

日時

平成23年10月26日(水曜日) 13時30分~

場所

(独)農業・食品産業技術総合研究機構 動物衛生研究所 管理棟2F 大会議室
茨城県つくば市観音台3-1-5 (交通案内/会場案内(建物5))
Tel.:029-838-7707(企画管理部 業務推進室 交流チーム)
Fax.:029-838-7907

 

今回はJICA研修員 イスロックさんによる発表です。
開催場所が 管理棟2階 大会議室 となっておりますのでご注意願います。
参加希望者の事前登録は不要、当日参加も可能です。多数のご来聴をお待ちしております。

-水曜会幹事-

内容

座長:中村 義男(動衛研)

Improvement of ITS1-PCR system by using FTAR cards and AmpdirectR Plus buffer for detection of Trypanosoma evansi infection. (20分)

○ Isrok Malikus Sufi 1, 2, Yoshio Nakamura 2 ( 1 Disease Investigation Center Subang, Ministry of Agriculture, Indonesia, 2 National Institute of Animal Health, Japan)

Trypanosoma evansi is one of the most widely distributed hemoparasites in Asia, and its infection of rumnants is listed up among 13 priority animal diseases in Indonesia. For epidemiological studies on T. evansi infection, internal transcribed spacer (ITS) 1 of the ribosomal DNA has been a target of PCR amplification, and a convenient PCR system with FTAR cards (Whatman) has been reported. However, this system requires a time-consuming step of washing DNA samples spotted on FTA punches, and actual sensitivity has not yet been determined. This study describes the improvement of ITS1-PCR by using a combination of FTAR cards and AmpdirectR Plus buffer (Shimadzu), special PCR buffer capable of inactivating inhibitory factors in biological samples. Parasite stock was inoculated into rats, and blood samples were collected from the rats developing high parasitemia as well as non-infected rats. Blood samples were frozen for later DNA extraction, and also spotted on to FTA cards. PCR amplifications were compared between two systems, extracted DNA samples with usual PCR buffer (ExTaqTM buffer, TaKaRa) (usual system) and non-washed FTA punch samples with Ampdirect Plus buffer (improved system). Optimal number of thermal cycling was 35 cycles in both systems. Both systems showed amplifications of ITS1 regions of T. evansi and T. brucei brucei with a size of 480 bp, T. theileri from cattle 400 bp, and T. theileri -like trypanosome from sika deer 340 bp. No amplifications were detected in samples containing Babesia ovata, Theileria orientalis, Anaplasma marginale, A. centrale and Mycoplasma wenyoni, and in non-infected samples in both systems. The detection limits were 10 and 100 parasites/µL blood in the usual and improved systems, respectively. The improved system was less sensitive than the usual system, however, still sensitive enough to apply to practical use, equivalent to the sensitivity of detecting one parasite in 20 fields of a blood film in microscope observation with x 400 magnification. Costs for sample preparation and PCR amplification were 575 yen and 397 yen for the usual and improved systems, respectively. These results indicated that the improved ITS1-PCR system using FTA cards and Ampdirect Plus buffer is more convenient and cost effective method for field survey on T. evansi infection.

法人番号 7050005005207