Touré F S, Kassambara L, Williams T, Millet P, Bain O, Georges A J, Egwang T G
Centre International de Recherches Médicales de Franceville, Gabon.
Am J Trop Med Hyg. 1998 Jul;59(1):144-9. doi: 10.4269/ajtmh.1998.59.144.
The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.
在盘尾丝虫病流行地区，制定控制策略至关重要，这在很大程度上依赖于对隐匿感染个体的准确识别。基于编码罗阿丝虫15-kD蛋白基因的重复3区（15r3）序列，开发了聚合酶链反应（PCR）和巢式聚合酶链反应（巢式PCR）。对来自隐匿感染受试者的20份血样和现场采集的无微丝蚴血症个体的30份血样进行了检测。初始PCR产物的大小为396个碱基对（bp）。当使用引物15r3(1)和15r3(2)进行30个循环的初始扩增时，20份隐匿感染个体中的3份以及30份无微丝蚴血症个体中的5份的PCR产物在电泳后用溴化乙锭染色可观察到。随后的Southern印迹和与特异性探针的杂交显示，20份隐匿感染样本中的19份以及30份无微丝蚴血症样本中的23份出现杂交，但印迹与X线胶片曝光两天后才出现。当进行巢式PCR时（产物大小 = 366 bp，引物15r3(3)和15r3(4)），初始PCR产物Southern杂交呈阳性的20份隐匿感染样本中的19份以及30份无微丝蚴血症样本中的23份经溴化乙锭染色呈强阳性。使用先前描述的相同探针对巢式PCR产物进行定性Southern印迹，在仅4小时的极短曝光时间后就证实了溴化乙锭染色结果。这些结果表明，巢式PCR扩增产物具有特异性，其检测隐匿性盘尾丝虫病的灵敏度为95%。这种方法对于在无需对PCR产物进行印迹和杂交的情况下对大量人群进行活动性盘尾丝虫病筛查具有重要前景。
