摘要
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 Pneumocystis jirovecii（Pj）specific DNA fragments from the non- PneumocystisPneumonia（non-PCP）patients＇ bronchial alveolar lavage fluid（BALF）samples by using the conventional PCR,nested PCR（n PCR）and quantitative PCR（qPCR）methods. Methods BALF samples were obtained from 50 confirmed non-PCP cases.Pj mitochondrial large subunit r RNA（mt LSUr RNA）gene was selected as target gene for conventional PCR（Mt-PCR）and nestPCR（Mt-n PCR）. Pj major surface glycoprotein（Msg）gene was selected as another target gene for conventional PCR（Msg-PCR）. Non-PCP patients＇ BALF samples were screened by Mt-PCR,Mt-n PCR,and Msg-PCR methods simultaneously. ThePCR screened positive samples were further studied by qPCR targeted mt LSUr RNA and Msg genes. Pj specific DNA fragmentcopy numbers were investigated in these non-PCP patients＇ BALF samples. Results In the 50 non-PCP patients＇ BALFsamples,the positive rates of Mt- n PCR,Mt- PCR and Msg- PCR were 56%（28/50）,36%（18/50）,and 26%（13/50）,respectively. Among these,there were 5 cases（10%）simultaneously positive in all of the three Mt-n PCR,Mt-PCR and Msg-PCR tests. There were 13 cases（26%）positive in any two of the three tests,18 cases（36%）positive in any one of the three tests,and 14 cases（28%）simultaneously negative in all of the three tests,respectively. Among the 36 positive cases tested by Mt-qPCR,all cases（100%）were in the level of 1,000 to 9,999 copies/μL. Among the 36 positive cases tested by Msg-qPCR,therewere 3 cases（8.3%）in the level of 100 to 999 copies/μL,16 cases（44.4%）in the level of 1,000 to 9,999 copies/μL,9 cases（25%）in the level of 10,000 to 99,999 copies/μL,and 8 cases（22.2%）in the level of 105copies/μL and more respectively. Inthe pairing qPCR tests,there were 28 cases（77.8%）whose Msg- qPCR copy numbers were greater than Mt- qPCR copynumber. Conclusion In the non-PCP BALF samples,positive rate of Pj was 72% by nucleic acid amplification tests. Th
出处
《中国热带医学》
CAS
2015年第10期1172-1176,共5页
China Tropical Medicine
基金
首都医科大学基础临床科研合作基金(No.14JL03)
关键词
耶氏肺孢子菌
非肺孢子菌肺炎
核酸扩增试验
支气管肺泡灌洗液
Pneumocystis jirovecii
Non-Pneumocystis pneumonia
Nucleic acid amplification tests
Bronchial alveolar lavage fluid
