通过环介导恒温扩增鉴定疟原虫品种
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By LabMedica International staff writers Posted on 01 Aug 2018 |
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图片：在环烯烃共聚物(COC)板上用感染疟原虫的红细胞做原位LAMP的方案原理图（图片蒙日本国立产业技术综合研究所惠赐）。
疟疾是有不同种类的疟原虫引起的一种传染病，据报道其中五种会感染人类。因为恶性疟原虫引起的疟疾最严重，致死率高，所以准确及时的诊断对于有效管理至关重要。
当前诊断疟疾的金标准是用显微镜检查吉姆萨染色的血液涂片。因为鉴定疟原虫品种是由显微镜医师人工搜寻感染了疟原虫的红细胞，所以寄生虫血症不严重或混合感染的情况常见人为误差导致的误诊。
日本国立产业技术综合研究所(www.aist.go.jp)的科学家在经过亲水处理的微孔板上对受感染的红细胞(iRBC)执行原位环介导恒温扩增(LAMP)，以在一块玻片上分析尽可能多的iRBC。用原位LAMP测定在细胞水平上鉴定疟原虫能被完成。科研小组使用了来自健康O型血献血者的新鲜红细胞，他感染了恶性疟原虫，其中60%处于环状期，超过20%处于滋养体期，不到20%处于裂殖体期。
将红细胞悬液——包括培养的恶性疟原虫、3D7株、被感染的红细胞——摊在环烯烃共聚物(COC)板表面，事先用日本东京莎姆克股份有限公司(www.samcointl.com)的SAMCO RIE系统（经过亲水处理）对表面做反应离子刻蚀处理，使之变得亲水，再搁置10分钟，让红细胞沉积在板表面。用RPMI 1640培养基洗板，它是几乎在整个板表面上形成的单层红细胞。然后用吹风机把板吹干。用福尔马林固定红细胞，继而用Triton X-100进行透化。然后，用地高辛(DIG)标记的dUTP和一组特异引物进行LAMP反应，扩增恶性疟原虫的18S rRNA基因。用荧光显微镜检可以检测到荧光阳性细胞，它们带有结合荧光素的抗DIG抗体，这样的细胞即为被感染的红细胞。
该研究的论文发表于2018年6月19日的《疟疾杂志》(Malaria Journal)。作者总结说，他们的研究显示，用原位LAMP在经过亲水处理的COC板上在单细胞水平鉴定疟原虫品种，可以极其灵敏、准确地诊断疟疾。上述成果将增强金标准方法诊断疟疾的效果。
日本国立产业技术综合研究所>>> www.aist.go.jp
莎姆克股份有限公司>>> www.samcointl.com
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