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目的利用常规PCR、巢式PCR(Nest PCR,n PCR)和定量PCR(Quantitative PCR,qPCR)方法,调查非肺孢子菌肺炎(Non-Pneumocystis pneumonia,non-PCP)患者支气管肺泡灌洗液(Bronchial alveolar lavage fluid,BALF)中耶氏肺孢子菌(Pneumocystis jirovecii,Pj)的检出情况。方法入选non-PCP患者50例留取BALF,分别以线粒体大亚基r RNA(Mi-tochondrial large subunit r RNA,mt LSUr RNA)为靶基因进行常规PCR(Mt-PCR)和巢氏PCR(Mt-nPCR),以主要表面糖蛋白(Major surface glycoprotein,Msg)为靶基因进行常规PCR(Msg-PCR),检测Mt-PCR,Mt-nPCR和Msg-PCR三种方法的Pj特异性核酸片段检出率,并对PCR检出为阳性的样本分别用以mt LSU r RNA和Msg为靶基因的qPCR来检测相应Pj核酸片段拷贝数。结果 50例BALF中,Mt-n PCR、Mt-PCR及Msg-PCR的检出率分别为56%(28/50)、36%(18/50)和26%(13/50),其中三种检测方法同时阳性5例(10%),任意两种方法阳性13例(26%),任意一种方法阳性18例(36%),三种方法均为阴性14例(28%)。36例PCR检测结果阳性的标本分别进行mtLSUrRNA定量PCR(Mt-qPCR)和Msg定量PCR(Msg-qPCR)检测,其中Mt-qPCR检测结果为:1 000拷贝/μL~9 999拷贝/μL有36例(100%);Msg-qPCR检测结果为:100拷贝/μL~999拷贝/μL有3例(8.3%),1 000拷贝/μL~9 999拷贝/μL有16例(44.4%),10 000拷贝/μL~99 999拷贝/μL有9例(25%),105拷贝/μL以上8例(22.2%)。36例配对检测qPCR样本中,Msg-qPCR拷贝数大于Mt-qPCR拷贝数样本28例(77.8%)。结论在non-PCP患者BALF中,Pj核酸扩增方法的检出率为72%,Mt-qPCR拷贝数主要位于103拷贝/μL数量级,Msg-qPCR拷贝数主要分布于103至105拷贝/μL。用核酸扩增方法在BALF中检测出Pj时,临床意义解读需要谨慎。
Objective To investigate the clinical significance of bronchial alveolar lavage fluid in patients with non-Pneumocystis pneumonia (non-PCP) using conventional PCR, nested PCR (nPCR) and quantitative PCR (qPCR) alveolar lavage fluid (BALF)), the detection of Pneumocystis jirovecii (Pj). Methods BALF was isolated from 50 patients with non-PCP, and the common PCR (Mt-PCR) and nested PCR (MtDNA) were performed using the mt LSUr RNA as the target gene. -nPCR), PCR (Msg-PCR) with major surface glycoprotein (Msg) as the target gene and Pj-specific nucleic acid fragment detected by Mt-PCR, Mt-nPCR and Msg- And the PCR-positive samples were used to detect the corresponding Pj nucleic acid fragment copy number using qPCR with mt LSU rRNA and Msg as target genes, respectively. Results The positive rates of Mt-n PCR, Mt-PCR and Msg-PCR in BALF were 56% (28/50), 36% (18/50) and 26% (13/50) There were 5 positive cases (10%), 13 positive cases (26%) by any two methods, 18 cases (36%) positive cases by any one method and 14 cases (28%) negative by any of the three methods. The results of Mt-qPCR assay showed that the number of Mt-qPCR was between 1 000 copies / μL and 9 999 copies / μL with 36 (mtLSUrRNA quantitative PCR (Mt-qPCR) and Msg-qPCR The results of Msg-qPCR showed that there were 3 cases (8.3%) of 100 copies / μL to 999 copies / μL, 16 cases (44.4%) of 1 000 copies / μL to 9 999 copies / μL, There were 9 cases (25%) with 000 copies / μL ~ 99,999 copies / μL and 8 cases (22.2%) with 105 copies / μL. Of the 36 matched pairs of qPCR samples, Msg-qPCR copy number was greater than that of Mt-qPCR copy number sample in 28 cases (77.8%). Conclusion The detection rate of Pj nucleic acid amplification method was 72% in the BALF of non-PCP patients. The copy number of Mt-qPCR mainly located in the order of 103 copies / μL. The copies of Msg-qPCR mainly distributed in the range of 103 to 105 copies / μL. When using nucleic acid amplification to detect Pj in BALF, careful interpretation of the clinical implications is needed.