Glare E M, Paton J C, Premier R R, Lawrence A J, Nisbet I T
Microbiology Department, Adelaide Children's Hospital, North Adelaide, South Australia.
J Clin Microbiol. 1990 Sep;28(9):1982-7. doi: 10.1128/jcm.28.9.1982-1987.1990.
A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19. This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B. pertussis tested. It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus. The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR). This procedure was then applied to the rapid (5-h) detection of B. pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis. The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF). All of the IF-positive samples were PCR positive, as were 63 of the samples from which B. pertussis was eventually cultured. Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay. However, 33 culture- and IF-negative specimens were positive by PCR assay. Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives.
通过使用载体pUC19，将来自百日咳博德特氏菌的一个串联重复的1046碱基对（bp）的ClaI DNA片段克隆到大肠杆菌中。该片段分离后，与所检测的所有100株百日咳博德特氏菌临床分离株的DNA都能强烈杂交。结果表明，它与支气管败血博德特氏菌的单拷贝序列具有同源性，但与副百日咳博德特氏菌没有同源性，并且不与包括密切相关的巴斯德氏菌属、产碱菌属和嗜血杆菌属成员在内的多种其他细菌的裂解物印迹杂交。对这个1046 bp的片段进行了测序，重复元件内一个153 bp区域两侧的互补合成寡核苷酸被用作引物，利用聚合酶链反应（PCR）对该区域进行特异性扩增。然后将该方法应用于从332名疑似百日咳儿童采集的鼻咽分泌物中快速（5小时）检测百日咳博德特氏菌。该检测在总共98个样本中得到阳性结果，相比之下，培养法检测出66个阳性样本，直接免疫荧光法（IF）检测出33个阳性样本。所有IF阳性样本的PCR检测也呈阳性，最终培养出百日咳博德特氏菌的样本中有63个PCR检测呈阳性。231个IF和培养检测均为阴性的标本，PCR检测也为阴性。然而，有33个培养和IF检测均为阴性的标本，PCR检测呈阳性。其中一些标本是从培养确诊的百日咳患者的密切接触者中采集的，是这些患者的随访标本，或者是来自有百日咳血清学证据的患者，因此可能是真阳性而非假阳性。
