Key words : influenza, nested-pcr, serotype
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1 Key words : influenza, nested-pcr, serotype
2 Table 1 Sequence of PCR primers used to detect HA gene of influenza virus
3 524 清水 Fig. 1 Detection of PCR product 英明 他 (lstpcr) Table 2 Results method Fig. 2 Detection B of PCR product B B primer,各 型 間 で の 交 差 反 応 は み られ ず プ ラ イ マ ー の 特 異 性 が 証 明 さ れ た. ン ドが 検 出 さ れ た(Table の検 出 感 度 の検 討 の 検 出 感 度 に つ い て プ ラ ー ク 法 を用 い 型 2ndPCRで0.9PFU/ は1stPCRで100PFU/assey ndpcrで1.opfu/assey,b型 PFU/assey, はl 2ndPCRで1 (78.9%)でDNAバ は1stPCRで180.8PFU/asseyで れ た(Table 4).合 との比 較 ウ イ ル ス 分 離 陽 性 の 咽 頭 ぬ ぐ い 液23件 は全 て分 離 ウ イ ル ス と 同 じ型 の プ ラ イ マ ー に の みDNAバ 計1stPCRで46検 た,PCR陽 ン フル エ ンザ 分 離 陰 性 で ペ ア血 清 で 抗 体 価 の 上 昇 が み られ は1stPCRで20検 件(5.0%),2ndPCRで17件(85.0%)でDNAバ 性 性 検 体 に つ い て抗 体 上 昇 PCR陽 ン ドが 増 幅 さ れ 性 は 発 病 後2 9日 体 中1 まで に 採 取 さ れ た 陽 性 で あ っ た7件 性 期 の 抗 体 価 が32倍 以 下 で あ っ た.ま はす べて 急 た,急 性期 の 抗 体 価 が32倍 以 下 か ら256倍 ま で,抗 体 価 の 比 較 的 高 い 検 体 か ら も,DNAバ 考 との 比 較 イ ン フ ル エ ン ザ 流 行 時 に 採 取 さ れ,イ 体 中7件.6%)がPCR陽 が み ら れ た 型 とPCRでDNAバ ン ドの 増 幅 が み ら れ た. ウ イ ル ス 分 離 陰 性 検 体 と.9%), ン ドが 検 出 さ (15.2%),2ndPCRで38件(82 血 清 で,1stPCRで ウ イ ル ス 分 離 陽 性 検 体 と 体 中3件(42 た 型 は全 て一 致 し た. あ っ た. た う が い 液 で は,型 ン ド が 検 出 さ れ た(Table で は1stPCRで7検 で あ っ た.ま,2 は1stPCR.8%),2ndPCRで15件 2ndPCRで6件(85.7%)でDNAバ 定 量 し た ウ イ ル ス で 検 討 を 行 っ た.型 assey, and PCR 2).型 体 中3件(15 3).B型 stpcrで90pfu/assey, titer type) (2ndPCR) で19検 2).1stPCR,2ndPCR共 of antibody ( 今 回,迅 ン ドが 検 出 さ れ た. 察 速 か つ 高 感 度 の 検 査 法 と し てNested- に よ る イ ン フ ル エ ン ザ ウ イ ル スRNAの 検 出 を ワ ク チ ン 株 を 用 い て 行 っ た と こ ろ,検 度 は 各 型 で,1stPCRで90 180PFU/assey, PCRで PFU/asseyで 2nd あ っ た.こ 感染症学雑誌 出感 第71巻 の こ と 第6号
4 Table 3 Results of antibody titer and PCR method ( type) Table 4 Results of antibody titer and PCR method (B type) 1) Goodman RA, Orenstein WA, Munro TF, Sikes KS : Impact of influenza A in a nursing home. JAMA 1982, 247 : ) Hall CB, Douglas RG : Nosocomial influenza infection as a cause of intercurrent fevers in infants. Pediatr 1975, 55 : 673.
5 12) Sugaya N, Kusumoto N, Suzuki Y, Nerome R, Nerome K : Large sequential outbreaks caused by influenza A(H3N2) and B viruses in an institution for the mentally handicapped. J 8) Yamada A, Imanishi J, Nakajima E, Nakajima Med Virol 1996 ; 50 : K, Nakajima S : Detection of influenza viruses in throat swab by using polymerase chain with influenza A infection. Treatment with 13) Hall CB, Dolin R, Gala CL et al. : Children reaction. Microbiol Immunol 1991 ; 35 : Rimantadine. Pediatr 1987 ; 80(2) : Rapid Detection of Influenza Virus A (, ) and B by Nested-Polymerase Chain Reaction Hideaki SHIMIZU1), SHIMIZU1, Sumi WATANABE & Mitsunobu IMAI2) Kawasaki City Institute of Public Health 2)Kanagawa Prefectural Public Health Laboratory We applied the Nested-polymerase chain reaction (PCR) for laboratory diagnosis of influenza virus infection. We used three primer sets for detection of influenza virus A (, ) and B. The primer sets for each type (,,B) was able to detect specifically each type of influenza. We measured the sensitivity for detection of vaccine strains. The PCR method was able to detect 0.9 PFU/assey of type, 1.0 PFU/assey of type and 1.8 PFU/assey of B type. Out of 46 isolation negative but antibody positive cases, 38 cases were positive for PCR (82.6%). This method is sensitive and useful for rapid diagnosis of influenza virus infection.
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