荧光PCR法半定量肺炎支原体的研究

Study on semi-quantitation of Mycoplasma pneumoniae by fluorescence quantitative PCR

目的：研究MPLI（肺炎支原体载量指数）的参考范围,探讨其在儿童肺炎支原体（MP）感染诊断中的应用价值。方法：2008年3月末采集3-6岁健康儿童咽拭子标本128例,采用FQ-PCR法分别检测MP-DNA和Hactin-DNA后计算MPLI值：MPLI=-lg（MP-DNA拷贝量/Hactin-DNA拷贝量）,然后算出MPLI的参考范围（P5）。2008年3月-7月于我院儿科采集临床患儿咽拭子标本110例及其双份血清,分别用FQ-PCR法和MP-IgM法检测咽拭子和血清标本。以单次或双次MP-IgM型抗体阳性作为判定MP感染的标准,算出各种方法的SEN、SPE、＋PV、-PV,并制作MPLI的ROC曲线。比较P5与ROC曲线所得界值的优劣。结果：ROC所得MPLI界值优于128例健康儿童MPLI的P5界值,所以,3-6岁儿童咽拭子MPLI的参考范围为：≥5.90。根据金标准,110例标本中有26例确诊为MP感染;以此为基础,一次MP-IgM法的SEN、SPE、＋PV、-PV分别为73.1%、100.0%、100.0%和92.3%;两次MP-IgM法的均为100.0%;单一FQ-PCR法的依次为26.9%、95.2%、63.6%和80.8%;MPLI的依次为26.9%、98.8%、87.5%和81.4%。结论：（1）单一FQ-PCR法测定MP-DNA的弱点很明显：假阳性率高,主要由于未对临床标本进行＂标准化＂,故而大大限制了其在临床上的大规模应用。（2）MPLI的优点在于对临床标本进行了＂标准化＂,充分弥补了单一FQ-PCR法假阳性率高的弱点,结果更具说服力;MPLI能早于MP-IgM法诊断MP急性感染。（3）在允许（如经济状况等）的情况下,MPLI和MP-IgM法的结合将会是很好的组合,既能早期发现MP急性感染,又能准确诊断MP感染的恢复（早）期状态。

Objective：To investigate the reference range of MPLI（Mycoplasma pneumoniae load index） and its usefulness in the diagnosis of Mycoplasma pneumoniae（MP） infection in children. Methods： 128 throat swabs were collected from 128 healthy children aged between 3 and 6 years at the end of March 2008. Fluorescence quantitative PCR（ FQ - PCR） assay was used to ana- lyze MP- DNA and human actin- DNA（ H actin -DNA） for those swabs. MPLI was then calculated： MPLI = -lg（ MP- DNA copies/H actin - DNA copies）, followed by the calculation of its reference rage（ P5 ）- 110 throat swabs were collected with paired sera from the department of pediatry in our hospital from March to July 2008. The former FQ - PCR assay was used to analyze throat swabs, MP - IgM assay for the sera. It was made a gold standard for MP infection that MP - IgM was positive once or twice, on which sensitivity（ SEN）, specificity（ SPE）, positive predictive value（ ＋ PV）, negative predictive value （-PV） and receiver operator characteristic curve （ROC） for MPLI were made based. P5 was compared with the cut -off from ROC. Results：The cut - off from ROC was better than that from P5 of 128 normal samples. Thus, the reference range of MPLI was determined as above 5.90 for children aged 3 to 6 years. 26 children were diagnosed MP infection among those 110 samples according to the gold standard. Then the values of SEN, SPE, ＋ PV, -PV were 73. 1%, 100.0%, 100.0% and 92.3% for a single MP- IgM, all 100. 0% for double MP - IgM respectively. Such were 26.9%, 95.2%, 63. 6% and 80. 8% for FQ - PCR alone, 26. 9%, 98. 8%, 87.5% and 81.4% for MPLI respectively. Conclusion： （1） FQ -PCR assay alone for MP- DNA is limited for its low SEN and high false positive rate, mainly due to non - standarization of clinical samples, which well restricts its clinical large - scale application. （2） The advantage of MPLI lies in its standarization of clinical samples, which makes up the limitation of high false positive rate for FQ - PCR alone and makes the results more persuasive. MPLI could help diagnose acute MP infection earlier than MP- IgM. （3） Under possible conditions（ such as economy）, MPLI and MP- DNA will be a good combination： it can not only detect acute MP infection earlier, but also help diagnose （early） convalescent state of MP infection.