荧光定量聚合酶链反应检测临床痰液标本中军团菌

Detection of Legionella in clinical sputum specimens by quantitative fluorescence-PCR

目的建立快速、特异、敏感的TaqMan探针荧光定量聚合酶链反应(PCR)方法,用于检测军团菌16SRNA基因。方法军团菌标准菌株购自美国标准菌种收藏所,临床痰标本取自南方医科大学南方医院257例病原不明肺炎住院患者。提取菌种DNA及临床痰标本DNA,通过对标准菌株及临床痰液标本DNA分别进行待考核试剂与16SrRNAPCR检测及测序,验证待考核试剂方法的敏感性、特异性。结果荧光定量PCR检测灵敏度为10cfu/mL,比16SrRNA定性PCR检测灵敏度381cfu/mL高38倍。257例病原不明肺炎患者的临床痰标本细菌培养检出嗜肺军团菌血清型1型1例。待考核试剂和16SrRNAPCR方法检测阳性率分别为8.95(23/257)和8.95(23/257),其中2例不符合,1例待考核试剂阳性16SrRNAPCR方法阴性,另一例16SrRNAPCR方法阳性,待考核试剂阴性。二者总符合率为99.22(255/257),其中阳性符合率为95.65(22/23),阴性符合率为99.57(233/234)。结论待考核试剂荧光定量PCR检测军团菌标准菌种敏感性强,特异性好,用于临床痰标本军团菌检测时假阳性率为4.35,与16SrRNAPCR方法及测序比较具有较好的符合性和等效性。

Objective To establish a rapid,specific and sensitive quantitative fluorescence--PCR assay using TaqMan probe for the detection of Legionella 16S rRNA gene. Methods Standard strain of LegioneIla was from the American Type Culture Col- lection （ATCC）. 257 cases of clinical sputum specimens were collected from patients with pneumonia of unknown pathogen in Nanfang Hospital,Southern Medical University. The genomic DNA of Legionella strain and clinical sputum specimens were extracted. The sensitivity and specificity of reagent to be assessed was detected by used for the detection of Legionella 16S rRNA in DNA samples from Legionella strain and clinical sputum specimens,and one commercialized 16S rRNA qualitation PCR reagent kit and DNA sequencing were used as controls. Results The sensitivity of quantitative fluorescence PCR was 10 cfu/mL,which was 38 times higher than that of qualitaion PCR reagent. One of the 257 cases of sputum specimens from patients with pneumonia of unknown pathogen was detected as Legionella pneumophila serotype 1 by bacterial culture. The positive rates of reagent to be assessed and qulitation PCR reagent were 8.95%（23/257） and 8.95 % （23/257） ,respectively. Two cases were not met. One case was positive, detected by reagents to be assessed, while negative detected by qualitaion PCR reagent. Another case was negative, detected by reagents to be assessed,while positive detected by qualitaion PCR reagent. The overall consistency rate was 99.22%0 （255/257）,the positive coincidence rate was 95.65% （22/23） and the negative coincidence rate was 99.57% （233/234）. Conclusion Reagent to be assessed, used to detect standard strain of Legionella, has good sensitivity and specificity. Its false positive rate is 4.35 % ,when it is used to detect clinical sputum specimens,and it has good consistency and equivalence compared with qualitation PCR reagent and DNA sequencing.