A POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF BRUGIA-MALAYI IN BLOOD
A POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION OF BRUGIA-MALAYI IN BLOOD
复制标题
DOI：
10.4269/ajtmh.1994.51.314
复制
发表时间：
1994-09-01
影响因子：
3.3
通讯作者：
WILLIAMS, SA
中科院分区：
文献类型：
--
作者：
LIZOTTE, MR;SUPALI, T;WILLIAMS, SA
由于传统的丝虫病诊断方法繁琐、耗时，且不能保证丝虫病的种属特异性，因此需要对丝虫病进行敏感、快速、种属特异性的诊断。一种基于聚合酶链反应（PCR）的检测方法，使用Hha I家族的高度重复的DNA序列从布鲁吉亚。在印度尼西亚的一项实地研究中收集了124份人类育雏样本，对该检测方法进行了测试。其中包括66例来自布氏丝虫病流行区患者的微丝蚴阳性样本，30例来自同一流行区的健康个体，28例来自非流行区的健康个体。在法属波利尼西亚的一个村庄收集了28个班氏吴策线虫病流行的幼虫样本，但没有B。马来人也进行了测试。使用传统的血涂片和膜过滤方法筛选血液样品，作为PCR检测的金标准。样品用蛋白酶K消化，苯酚和氯仿提取，透析。透析产物的一部分用于使用Hha I特异性引物的PCR。PCR检测正确地确定了所有的微丝蚴阳性样本为PCR阳性和所有的非流行性样本为PCR阴性。此外，30份来自流行区健康个体的样本中，26份也被鉴定为PCR阴性，4份PCR阳性。很可能这四个人有非常低水平的或隐蔽的感染，PCR检测检测循环DNA从死丝虫释放。结果表明，Hha Ⅰ PCR检测体系具有快速、特异、灵敏的特点。
There is need for sensitive, rapid, species-specific diagnosis of Brugia filarial parasites because traditional methods are tedious and time-consuming, with little guarantee of species specificity. A polymerase chain reaction (PCR)-based assay was developed using the Hha I family of highly repeated DNA sequences from Brugia. The assay was tested on 124 human brood samples collected in a field study in Indonesia. These included 66 microfilaria-positive samples from patients in an area endemic for Brugia, 30 from healthy individuals from the same endemic area, and 28 from healthy individuals from a nonendemic area. Twenty-eight brood samples collected in a village in French Polynesia endemic for Wuchereria bancrofti, but not B. malayi, were also tested. The blood samples were screened using the traditional blood smear and membrane filtration methods, which served as the gold standards to which the PCR assay was compared. The samples were digested with proteinase K, extracted with phenol and chloroform, and dialyzed. A fraction of the dialyzed product was used in PCRs using Hha I-specific primers. The PCR assay correctly identified all of the microfilaria-positive samples as PCR positive and all of the nonendemic samples as PCR negative. Additionally, 26 of 30 samples from healthy individuals in the endemic area were also identified as PCR negative, while four were PCR positive. It is likely that these four individuals had very low-level or cryptic infections, and that the PCR assay detected circulating DNA released from dead filariae. The results indicate that the Hha I PCR detection system is rapid, species-specific, and sensitive.