Oliveira D A, Holloway B P, Durigon E L, Collins W E, Lal A A
Immunology Branch, National Center for Infectious Diseases, Atlanta, Georgia.
Am J Trop Med Hyg. 1995 Feb;52(2):139-44. doi: 10.4269/ajtmh.1995.52.139.
In the present study, we describe a polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay for the detection of malaria infection. The target region of the 18S ribosomal DNA is amplified by a PCR using an 18S rRNA, genus-specific, biotinylated (5') and an unlabeled primer (3') pair. The detection probes are digoxigenin-labeled DNA oligonucleotides derived from species-specific rRNA sequences. The amplified fragments are allowed to hybridize with the species-specific, digoxigenin-labeled oligonucleotide probes. The oligo/DNA complex is allowed to bind onto streptavidin-coated microtiter plates, followed by incubation with a peroxidase-streptavidin conjugate and a colorimetric-peroxidase substrate. The resulting test demonstrated specificity for the four human Plasmodium species, and was able to detect a level of parasitemia of at least 0.0001% in a laboratory-induced P. falciparum infection in monkeys. This liquid hybridization assay is sensitive, specific, simple, and reliable, with wide applicability in epidemiologic studies, accurate detection of mixed infections, detection of low-level parasitemia, and evaluation of chemotherapy and vaccine efficacy.
在本研究中，我们描述了一种基于聚合酶链反应（PCR）的酶联免疫吸附测定法，用于检测疟疾感染。使用生物素化的（5'）18S rRNA属特异性引物和未标记的（3'）引物对，通过PCR扩增18S核糖体DNA的目标区域。检测探针是源自种特异性rRNA序列的地高辛配基标记的DNA寡核苷酸。使扩增的片段与种特异性的、地高辛配基标记的寡核苷酸探针杂交。使寡核苷酸/DNA复合物结合到链霉抗生物素蛋白包被的微量滴定板上，随后与过氧化物酶-链霉抗生物素蛋白缀合物和比色过氧化物酶底物一起孵育。所得试验对四种人类疟原虫具有特异性，并且能够在实验室诱导的猴恶性疟原虫感染中检测到至少0.0001%的寄生虫血症水平。这种液相杂交测定法灵敏、特异、简单且可靠，在流行病学研究中具有广泛的适用性，可准确检测混合感染、检测低水平寄生虫血症以及评估化疗和疫苗效力。
