Goodman J L, Jurkovich P, Kramber J M, Johnson R C
Department of Medicine, University of Minnesota School of Medicine, Minneapolis 55455.
Infect Immun. 1991 Jan;59(1):269-78. doi: 10.1128/iai.59.1.269-278.1991.
Current diagnostic tests for Lyme disease (LD) are dependent upon the host serologic response and are insensitive early in infection and, possibly, following antibiotic therapy. We cloned a library of Borrelia burgdorferi 297 DNA and studied one clone, Ly-1, for its potential in diagnostic and pathogenic studies. Using pulsed-field electrophoresis, we demonstrated that Ly-1 is of chromosomal origin and estimated that the B. burgdorferi chromosome is approximately 1,100 kb in size. The 3.7-kb Ly-1 clone hybridizes with geographically diverse strains of B. burgdorferi. No cross hybridization occurs with DNA from human cells, Escherichia coli, Staphylococcus aureus, Clostridium difficile, or the closely related B. hermsii. We used a dot blot assay to detect 100 pg of B. burgdorferi DNA. We partially determined the nucleotide sequence of Ly-1 and used it to select and synthesize oligonucleotides for use in the polymerase chain reaction (PCR). Two different primer pairs were found to amplify DNA from nine geographically diverse isolates. We could detect 10 fg (less than 10 molecules) of B. burgdorferi or less than five spirochetes added to human urine. Finally, we were able to use the PCR to detect B. burgdorferi DNA in the urine of four of eight patients with suspected active LD (three with arthritis and one with neurologic manifestations), all of whom responded to antibiotic treatment. In contrast, those patients who were PCR negative either had inactive disease or had been appropriately treated and did not respond to additional antibiotics, and all four control urine specimens were PCR negative. We conclude that B. burgdorferi DNA can be sensitively detected by the PCR with the primers and methods we describe and that the urinary tract is a site of persistent infection in some cases of human LD, an observation of potential diagnostic and pathogenic importance.
目前莱姆病（LD）的诊断测试依赖于宿主的血清学反应，在感染早期以及可能在抗生素治疗后都不敏感。我们克隆了伯氏疏螺旋体297株的DNA文库，并研究了其中一个克隆Ly-1在诊断和致病研究中的潜力。使用脉冲场凝胶电泳，我们证明Ly-1来自染色体，并估计伯氏疏螺旋体染色体大小约为1100 kb。3.7 kb的Ly-1克隆与来自不同地理区域的伯氏疏螺旋体菌株杂交。与人细胞、大肠杆菌、金黄色葡萄球菌、艰难梭菌或密切相关的赫氏疏螺旋体的DNA没有交叉杂交。我们使用斑点印迹法检测100 pg的伯氏疏螺旋体DNA。我们部分测定了Ly-1的核苷酸序列，并用于选择和合成用于聚合酶链反应（PCR）的寡核苷酸。发现两对不同的引物可扩增来自九个不同地理区域分离株的DNA。我们能够检测到添加到人类尿液中的10 fg（少于10个分子）的伯氏疏螺旋体或少于五个螺旋体。最后，我们能够使用PCR检测八名疑似活动性LD患者中的四名患者尿液中的伯氏疏螺旋体DNA（三名患有关节炎，一名有神经学表现），所有这些患者对抗生素治疗均有反应。相比之下，那些PCR阴性的患者要么患有非活动性疾病，要么已接受适当治疗且对额外抗生素无反应，并且所有四个对照尿液标本PCR均为阴性。我们得出结论，使用我们描述的引物和方法，通过PCR可以灵敏地检测到伯氏疏螺旋体DNA，并且在某些人类LD病例中，泌尿道是持续感染的部位，这一观察结果具有潜在的诊断和致病重要性。
