Rao U R, Williams S A, Klei T R
Department of Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA.
Mol Cell Probes. 2002 Feb;16(1):13-23. doi: 10.1006/mcpr.2001.0393.
The sensitive, rapid and species-specific diagnosis of Brugia infections in humans or animal models is important in determining the level of parasitemia and the efficacy of chemotherapy or vaccinations. The HhaI family of highly repeated DNA sequences from Brugia have been useful in polymerase chain reaction (PCR)-based diagnosis of brugian filarial infections in blood samples and in mosquitoes. A PCR assay was developed using a biotinylated primer, a non-biotinylated primer and a species-specific chemiluminescent probe [tris[2,2'bipyridine] ruthenium (II) chelate, TBR] to detect PCR amplified Hhal family repeats. Individual blood samples from jirds infected with Brugia malayi or B. pahangi and with different levels of microfilaremia were tested in this assay. Known concentrations of Brugia DNA and DNA from the blood of uninfected control jirds were used as positive and negative controls, respectively. The PCR products generated by this method were analyzed using a semi-automated quantitative (Q)-PCR system. The levels of parasite DNA can be calculated from the luminosity units generated. Significant amounts of parasite DNA were detected in blood samples from infected jirds, and these values were correlated with the levels of microfilaremia. In contrast, reductions in circulating microfilaria following treatment with ivermectin correlated with low levels of measurable DNA. Using this system, we were also able to detect HhaI repeat DNA in the spleens of B. pahangi- infected jirds at 56 days post-infection when circulating microfilariae were not readily detectable. The results indicate that the species-specific Hhal Q-PCR detection and quantification method is rapid and sensitive, is useful in the detection of Brugia DNA in blood and other tissues and is suited for use in clinical settings because it does not require radioactive isotopes and gel-based protocols.
对人类或动物模型中的布鲁氏菌感染进行灵敏、快速且具有种属特异性的诊断，对于确定寄生虫血症水平以及化疗或疫苗接种的疗效至关重要。来自布鲁氏菌的高度重复DNA序列的HhaI家族，在基于聚合酶链反应（PCR）的血液样本和蚊子中布鲁氏丝状虫感染诊断中很有用。利用生物素化引物、非生物素化引物和种属特异性化学发光探针[三（2,2'-联吡啶）钌（II）螯合物，TBR]开发了一种PCR检测方法，以检测PCR扩增的Hhal家族重复序列。在此检测中，对感染马来布鲁氏菌或彭亨布鲁氏菌且微丝蚴血症水平不同的沙鼠的个体血液样本进行了检测。已知浓度的布鲁氏菌DNA和未感染对照沙鼠血液中的DNA分别用作阳性和阴性对照。使用半自动定量（Q）-PCR系统分析此方法产生的PCR产物。可根据产生的发光单位计算寄生虫DNA的水平。在感染沙鼠的血液样本中检测到大量寄生虫DNA，这些值与微丝蚴血症水平相关。相比之下，用伊维菌素治疗后循环微丝蚴的减少与可测量DNA的低水平相关。使用该系统，我们还能够在感染彭亨布鲁氏菌的沙鼠感染后56天，在脾脏中检测到HhaI重复DNA，此时循环微丝蚴不易检测到。结果表明，种属特异性Hhal Q-PCR检测和定量方法快速、灵敏，可用于检测血液和其他组织中的布鲁氏菌DNA，且由于不需要放射性同位素和基于凝胶的方案，适用于临床环境。
