LUMINEX-MPMA和NGS技术在中枢神经系统感染性疾病诊断中的应用研究

发布时间：2019-06-12 20:37

【摘要】：中枢神经系统感染性疾病具有进展迅速,致死致残率高等特征。导致中枢神经系统感染的病原体种类达100多种,包括病毒,细菌,真菌,寄生虫等。但目前的病原体检测方法(如镜检、培养、ELISA检测、生化、PCR检测等)阳性率低、诊断周期长,且无法达到高通量检测病原体的要求。由于缺乏高通量、阳性率高的诊断方法,60%-85%的患者无明确病原学诊断[1],患者被给予经验型治疗,不仅导致了抗生素的滥用及微生物的耐药,并且给患者带来了沉重的经济负担。因此,为有效降低该类疾病的不良影响,在临床上对导致疾病的病原体进行高特异性、高通量、快速的检测的方法是迫切需要的。本研究利用Luminex TM-200和NGS技术,以疑诊病毒性脑(膜)炎患者脑脊液为标本,建立能同时筛查18种病原体的Luminex TM-200平台及探索性研究二代测序分析技术能否应用于脑脊液标本检测并发现未知病原体。第一部分基于LuminexTM-200平台高通量检测18种病原体方法的建立目的:通过LuminexTM-200平台建立一种通量高、特异性强的可同时检测18种中枢神经系统常见病原体的核酸检测平台。方法:建立基于Luminex TM-200为平台的MPMA-PCR体系,该体系应用Luminex磁珠微阵列标记,多重PCR及温度转移扩增等三种策略,可同时扩增18种病原体(hsv-1,hsv-2,vzv,cmv,eb,mev,muv,jcv,jev,echo,ev71,ca16,hev,c.neoforman,m.tuberculosis,s.pneumonia,t.gondii,a.cantonensis)。首先,针对18种病原体的特异性基因序列构建质粒并作为标准品,对标准品检测的基础之上建立mpma-pcr体系并以pcr产物测序结果作为金标准,验证mpma-pcr体系的特异性。通过检测倍比稀释的标准品检测mpma-pcr体系95%最低检出浓度。其次,收集177例脑脊液样本(包括138例疑诊病毒性脑(膜)炎脑脊液样本,19例经商业化试剂盒检测的脑脊液样本,20例非脑(膜)炎脑脊液样本),使用mpma-pcr体系进行检测,并以测序结果评价该体系检测临床样本的可行性、特异性及灵敏度。结果:(1)mpma-pcr体系的建立:通过标准品质粒的检测,确认mpma-pcr体系可针对18种病原体特异性基因同时检测。mpma-pcr体系检测结果与测序验证实验结果一致,特异性为100%。应用probit模型分析mpma-pcr各引物的95%最低检出限hsv-2、vzv、eb和muv的引物的95%最低检出限为103copy/reaction。其余14对引物95%最低检出限均高于100copy/reaction。(2)临床样本的检测:应用mpma-pcr体系检测177例脑脊液样本病原核酸阳性率23.16%(41/177):hsv-1病毒检出率为7.9%(14/177);hsv-2病毒检出率6.21%(11/177);eb病毒和埃可病毒检出率为2.25%(4/177);巨细胞病毒和带状疱疹病毒各2例;腮腺炎病毒、ca16、ev71和ev68各1例。阴性对照样本,未检测到病原体。mpma-pcr体系检测结果与测序验证结果达到95.48%的一致性。结论:建立一种基于luminextm-200平台高通量、高特异性的mpma-pcr体系,该体系可同时检测18种中枢神经系统感染病原体。mpma-pcr体系检测标准品检测结果与测序验证达到100%的符合率。通过对临床疑诊病毒性脑(膜)炎患者脑脊液标本检测,mpma-pcr体系病原体检出率可达23.16%,特异性达到100%。针对检出率较低,我们主要考虑两方面原因,第一,导致出现类似病毒性脑膜炎症状的原因可能超出mpma-pcr体系目标检测范围。第二,疑似脑(膜)炎的病例,可能由于致病病原体数量少,才导致症状不典型,且核酸含量低于mpma-pcr检测的最低检测浓度。综合以上原因,mpma-pcr体系,灵敏度并不太理想。但是结合金标准比对结果显示,该体系具有较高的特异性。第二部分 NGS技术在中枢神经系统感染未知病原体检测的探索性研究目的:实验第一部分结果显示,MPMA-PCR体系具有较高的特异性,但是灵敏度缺乏。仍有部分脑脊液样本病原体信息不能明确,为进一步明确这些样本的病原体信息,我们探索性应用灵敏度更高的二代测序平台(NGS),以期发现其它可能导致中枢神经系统感染的病原体。方法:收集经第一部分实验仍无明确病原体信息的脑脊液样本21例(为未知病源组)和12例明确诊断为隐球菌脑膜炎患者脑脊液样本(为隐球菌组)作为阳性对照,利用NGS技术对33例未知病原体样本检测进行探索性研究;NGS阳性结果利用qPCR方法进行验证。结果:12例隐球菌脑膜炎样本经NGS检测,83.33%(10/12)样本中存在隐球菌序列。经qPCR平行验证阳性率为33.33%(4/12);21例未知病原体样本中,28.57%(5/21)高度可能病原菌包括:克雷伯杆菌1例,肺炎链球菌1例,无乳链球菌1例,结核杆菌1例,假单胞菌1例;14.28%(3/21)的样本经qPCR确认样本(包括1例克雷伯杆菌,1例肺炎链球菌,1例无乳链球菌)。NGS在本次脑脊液样本建库成功率仅为72%(24/33)。结论:针对隐球菌阳性对照样本检测,83.33%的样本中存在隐球菌序列,但qPCR验证检测仅33.33%的样本检出隐球菌。这一结果表明根据隐球菌组二代测序技术的灵敏度高于qPCR。根据阳性对照样本的NGS检测结果表明,NGS技术针对脑脊液中病原体核酸检测具有可行性,且高灵敏度。经NGS技术检测未知病原体组,28.57%的样本可检测到高度怀疑的病原体序列。经qPCR验证得到阳性率为14.28%。21例未知病原体样本中检测到其他病原体核酸,表明了NGS技术对MPMA-PCR体系灵敏度的补充。但是目前72%的建库成功率相对较低,建库的成功了将直接影响NGS技术的灵敏度。但是目前NGS平台针对脑脊液标本检测并没有成熟的文库建立方法,所以如何解决病原体核酸载量低的问题尚需更多实验研究和探索。
[Abstract]:The infectious diseases of the central nervous system have the characteristics of rapid progress, high death rate and the like. The types of pathogens that lead to the infection of the central nervous system are more than 100, including viruses, bacteria, fungi, parasites, and the like. But the current pathogen detection method (such as microscopic examination, culture, ELISA detection, biochemistry, PCR detection, etc.) has the advantages of low positive rate, long diagnosis period and no requirement of high-flux detection of the pathogen. Due to the lack of high flux and high positive rate of the diagnosis,60% to 85% of the patients have no clear etiological diagnosis[1], and the patient is treated with an empirical type, which not only leads to the abuse of the antibiotics and the drug resistance of the microorganisms, but also brings a heavy economic burden to the patients. Therefore, in order to effectively reduce the adverse effect of the disease, the method of high specificity, high-throughput and rapid detection of the pathogen causing the disease is urgently needed. Luminex (TM-200) and NGS (NGS) technology were used in this study to establish a Luminex TM-200 platform and an exploratory study on the screening of 18 pathogens. The first part is based on the aim of high-throughput detection of 18 pathogen methods based on the Luminex TM-200 platform: a nucleic acid detection platform with high flux and strong specificity can be established through the Luminox TM-200 platform, and the common pathogens of the 18 central nervous systems can be detected at the same time. Methods: An MPMA-PCR system based on Luminex TM-200 as a platform was established. The system applied the three strategies of Luminex magnetic bead microarray labeling, multiplex PCR and temperature transfer amplification, and can simultaneously amplify 18 pathogens (hsv-1, hsv-2, vzv, cmv, eb, mev, muv, jcv, jv, echo, ev71, ca16, hev, c. neoforman, m. tubulosis, s. pneumonia, T. gondii, a. canonensis). First, a plasmid was constructed for the specific gene sequence of 18 pathogens and used as the standard, and the mpma-pcr system was established on the basis of the standard product detection and the result of the sequencing of the pcr product was used as the gold standard to verify the specificity of the mpma-pcr system. The minimum detectable concentration of the mpma-pcr system was detected by the test of the standard product diluted in comparison to the dilution. Then,177 cerebrospinal fluid samples (including 138 cerebrospinal fluid samples of suspected viral encephalitis (membrane),19 cerebrospinal fluid samples detected by the commercial kit and 20 non-brain (membrane) inflammatory cerebrospinal fluid samples) were collected and tested using the mpma-pcr system. The feasibility, specificity and sensitivity of the system were evaluated by sequencing. Results: (1) The establishment of the mpma-pcr system: the detection of the standard quality particles confirmed that the mpma-pcr system can be used for simultaneous detection of 18 pathogen-specific genes. The results of the mpma-pcr system were consistent with the results of the sequencing validation, and the specificity was 100%. The detection limit of the detection limit of 95% of the primers of the mma-pcr primers was analyzed by using the probit model, and the detection limit of the 95% of the primers for the vzv, b and muv was 103 copy/ action. The minimum detection limit of the remaining 14 pairs of primers was higher than 100 copy/ reaction. (2) The detection of clinical samples: The positive rate of pathogenic nucleic acid in 177 patients with cerebrospinal fluid sample was 23.16% (41/177) by using the mpma-pcr system. The detection rate of hsov-1 virus was 7.9% (14/177), the detection rate of hsov-2 virus was 6.21% (11/177), the detection rate of eb virus and Esmovirus was 2.25% (4/177),2 cases of cytomegalovirus and herpes zoster virus, and ca16, 1 case of ev71 and ev68. Negative control sample, no pathogen detected. The results of the detection of the mpma-pcr system and the results of the sequencing verification reached 95.48%. Conclusion: A high-flux and high-specificity mpma-pcr system based on luminextm-200 is established, which can detect the pathogens of 18 central nervous systems at the same time. The results of the detection of the mma-pcr system and the sequencing verification reached 100%. By means of the detection of cerebrospinal fluid in the patients with viral encephalitis (membrane), the detection rate of the mma-pcr system can reach 23.16%, and the specificity can reach 100%. In view of the low detection rate, we mainly consider two reasons, first, the cause of similar viral meningitis symptoms may exceed the target detection range of the mpma-pcr system. Second, cases of suspected brain (membrane) inflammation may cause the symptoms to be not typical due to the small number of pathogenic agents, and the nucleic acid content is lower than the lowest detection concentration detected by the mpma-pcr. For the above reasons, the mpma-pcr system is not ideal for sensitivity. However, the results show that the system has a higher specificity than the gold standard. The first part of the experiment shows that the MPMA-PCR system has a high specificity, but the sensitivity is lacking. There are still some of the cerebrospinal fluid sample pathogen information that is not clear, and in order to further clarify the pathogen information for these samples, we have an exploratory application of the second-generation sequencing platform (NGS) with a higher sensitivity, with a view to finding other pathogens that may lead to the central nervous system infection. Methods: The cerebrospinal fluid samples (for unknown source group) and 12 cases of the cerebrospinal fluid sample of cryptococcal meningitis were collected as positive control by the first part of the experiment. An exploratory study of 33 unknown pathogen samples was performed using NGS technique, and the NGS positive results were verified by qPCR. Results:12 cases of cryptococcal meningitis were detected by NGS and the sequence of cryptococcus was found in 83.33% (10/12) samples. The positive rate of qPCR was 33.33% (4/12); in 21 unknown pathogen samples, 28.57% (5/21) of the highly probable pathogenic bacteria included:1 of Klebsiella,1 in Streptococcus pneumoniae,1 in non-dairy streptococcus,1 in Mycobacterium tuberculosis and 1 in Pseudomonas; A sample of 14.28% (3/21) was confirmed by qPCR (including 1 Klebsiella,1 Streptococcus pneumoniae,1 non-Streptococcus). The success rate of NGS in this CSF sample was only 72% (24/33). Conclusion: Cryptococcus neoformans were found in 83.33% of samples for cryptococcus positive control samples, but only 33.33% of the samples were detected by qPCR. The results showed that the sensitivity of the second generation sequencing technique was higher than that of qPCR. The results of NGS test according to the positive control sample show that the NGS technique is feasible and highly sensitive to the detection of the pathogen nucleic acid in the cerebrospinal fluid. The unknown pathogen group was detected by the NGS technique, and 28.57% of the samples were able to detect highly suspected pathogen sequences. The positive rate was 14.28% by qPCR, and the other pathogen nucleic acid was detected in 21 unknown pathogen samples, which indicated that the NGS technique was complementary to the sensitivity of the MPMA-PCR system. However, at present,72% of the construction success rate is relatively low, and the success of the building will directly affect the sensitivity of the NGS technology. However, at present, the NGS platform has no mature library establishment method for cerebrospinal fluid specimen detection, so it is necessary to study and explore the problem of how to solve the problem of low pathogen nucleic acid load.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R741

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