双重聚合酶链反应法早期诊断军团菌肺炎的初步研究

A pilot study on the value of duplex polymerase chain reaction method in early diagnosis of Legionella pneumonia

目的 探讨双重聚合酶链反应 (DPCR)法检测痰及支气管肺泡灌洗液 (BALF)中军团菌DNA在早期诊断军团菌肺炎的意义。方法 用DPCR对军团菌肺炎组 (15例 )和普通肺炎组 (31例 )患者病程早期留取的痰及BALF进行军团菌DNA检测。用军团菌 16SrRNA基因和mip基因序列引物 ,可扩增 386bp 16SrRNA基因片段和 2 0 6bpmip基因片段。通过模拟标本来检测DPCR方法用于临床标本时的灵敏度。结果 军团菌肺炎组患者痰、BALF的DPCR结果均为阳性 ,其中 13例患者留取的 2 3份痰、7份BALF标本 386bp 16SrRNA基因片段和 2 0 6bpmip基因片段的扩增结果均阳性 ,余 2例患者留取的 2份痰和 1份BALF标本单一 386bp基因片段扩增阳性。DPCR结果与血清军团菌抗体结果相符。普通肺炎组患者痰和BALF的DPCR结果均为阴性。同一患者留取的多份标本呈现相同的DPCR结果。模拟痰和BALF的DPCR最低检出限均为 1× 10 3 cfu/ml。结论 采用DPCR法检测痰和BALF中的军团菌DNA ,具有较好的敏感性、特异性和稳定性 。

Objective To investigate the value of duplex polymerase chain reaction (DPCR) in early diagnosis of Legionella pneumonia by detecting Legionella DNA in sputum and bronchoalvelar lavage fluid(BALF). Methods During the process of DPCR, two different sets of oligonucleotide primers were stimultaneously used to amplify 386bp 16SrRNA gene fragment and 206bp mip gene fragment. These two primers were designed according to the sequences of 16SrRNA gene and mip gene of Legionella. The sputum and BALF of patients from two groups, including a Legionella pneumonia group ( n =15) and an ordinary pneumonia group( n =31) were collected at early course of the disease. All of the samples were detected with DPCR for Legionella DNA. Simulated samples were also detected to investigate the sensitivity of the method for testing clinical samples. Results All the samples collected from the Legionella pneumonia patients, including 25 of sputum,and 8 of BALF showed positive DPCR. The results of DPCR, which could distinguish Legionella pneumophila from non pneumophila Legionella spp. to some degree, were in good accordance with those of the specific serum antibodies. All of the samples from the ordinary pneumonia group including 40 of sputum and 16 of BALF demonstrated negative DPCR.Different samples of the same patient showed the same DPCR results. The lowest detection level of simulated sputum sample was the same as that of simulated BALF sample, being 1×10 3 cfu/ml. Conclusions This preliminary study showed that DPCR had satisfactory sensitivity, specificity and stability for detecting Legionella DNA in sputum and BALF. The method is of value in early diagnosis of Legionella pneumonia. Its wide use for clinical work requires further investigation.