LAMP法を利用した導電性中空糸膜による模擬血清試料からのウイルス検出

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Human blood collected from donors is tested for viruses using nucleic acid amplification testing (NAT) because blood products are made of human blood in Japan. However, NAT cannot detect viruses if there is only slight contamination. Therefore, viral infection is often caused by blood products contaminated with viruses. To prevent viral infection through blood products, we designed a viral detection method that uses a metal-coated hollow fiber (MCH)-membrane coated with gold. The principle of this viral detection is as follows. Viruses are captured and concentrated in a MCH-membrane, a viral sample is aspirated through the membrane. The membrane is then soaked in an alkaline-sodium dodecyl sulfate (SDS)-solution, and viruses are chemically lysed. After that, the membrane is used as a cathode electrode. Viral genes are electrically released from the membrane to electrophoresis buffer when a 3.5 V/cm electric field is applied for 5 min. Viruses are then detected by amplifying the released genes. Our aim is realize clinical application for viral detection, and we demonstrated the system on simulated serum samples for the first time. In addition, we introduce loop-mediated isothermal amplification (LAMP) as a method of nucleic acid amplification and attempt to shorten the detection time. The viral detection method using MCH-membrane was not inhibited by the proteins contained in calf serum. Viruses were detected from simulated serum samples containing 10 PFU/mL of herpes simplex virus type 1 (HSV-1) with this viral detection method. The detectable sensitivity was caused by the viral concentration of MCH-membrane. Because LAMP markedly shortens the detection time, viral detection was completed in approximately one-fourth the detection time of NAT (90 min). Viral detection using MCH-membrane will contribute to not only the inhibition of viral infection with contaminated blood products, but also to the rapid provision of platelet products, which have short expiration dates.
2004-12-10

LAMP法を利用した導電性中空糸膜による模擬血清試料からのウイルス検出

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