Toure F S, Egwang T G, Wahl G, Millet P, Bain O, Georges A J
Centre International de Recherches Medicales de Franceville, Franceville, Gabon.
Am J Trop Med Hyg. 1997 Jan;56(1):57-60. doi: 10.4269/ajtmh.1997.56.57.
A polymerase chain reaction (PCR)-based method to detect Loa loa DNA in the blood lysate of infected individuals is described. A set of primers was designed to amplify the repeat 3 sequence (15r3) of the gene encoding a putative L. loa allergen. The qualitative PCR was carried out using blood lysates from subjects from an L. loaendemic area of Gabon where loiasis exists sympatrically with Mansonella perstans, and from individuals from a loiasis-free area in Togo infected concomitantly with M. perstans and Onchocerca volvulus. No specific amplification was observed after ethidium bromide staining of a gel containing M. perstans and O. volvulus control samples. In contrast, a 396-basepair (bp) DNA was detected in all L. loa microfilaremic individuals and in seven of the 20 L. loa amicrofilaremic subjects diagnosed by leukoconcentration. Qualitative Southern blots carried out at high stringency (65 degrees C) using 15r3 oligonucleotide probe revealed hybridization only with L. loa samples (5 of 5 microfilaremic individuals and 15 of 20 amicrofilaremic individuals), confirming the results obtained with ethidium bromide staining of PCR products. We conclude that this 396-bp sequence could be used as a species-specific diagnostic tool for occult loiasis in an endemic area with concurrent filarial infections.
本文描述了一种基于聚合酶链反应（PCR）的方法，用于检测感染个体血液裂解物中的罗阿丝虫DNA。设计了一组引物，以扩增编码假定的罗阿丝虫变应原的基因的重复3序列（15r3）。使用来自加蓬罗阿丝虫流行地区（罗阿丝虫病与常现曼森线虫共存）的受试者的血液裂解物，以及来自多哥无罗阿丝虫病地区、同时感染常现曼森线虫和盘尾丝虫的个体的血液裂解物进行定性PCR。对含有常现曼森线虫和盘尾丝虫对照样品的凝胶进行溴化乙锭染色后，未观察到特异性扩增。相反，在所有罗阿丝虫微丝蚴血症个体以及通过白细胞浓缩法诊断的20名罗阿丝虫无微丝蚴血症受试者中的7名中检测到了一条396个碱基对（bp）的DNA。使用15r3寡核苷酸探针在高严谨度（65℃）下进行的定性Southern印迹显示，仅与罗阿丝虫样品杂交（5名微丝蚴血症个体中的5名和20名无微丝蚴血症个体中的15名），证实了PCR产物溴化乙锭染色获得的结果。我们得出结论，这条396-bp序列可作为在同时存在丝虫感染的流行地区隐匿性罗阿丝虫病的种特异性诊断工具。
