IR@PKUHSC  > 北京大学航天临床医学院
学科主题临床检验诊断学
血流常见真菌感染实时荧光定量PCR快速检测平台的建立及其应用研究
郭毅
2016-05-18
导师梁国威
专业内科学
授予单位北京大学
授予地点北京大学航天临床医学院
学位硕士
关键词念珠菌 荧光定量PCR 5.8S rRNA 基因 内转录间隔序列 全血标本
其他题名Real-Time PCR Assays for Rapid Detection and Identification of Common Fungi from Whole Blood Samples
分类号R446.5
摘要

背景:临床真菌引起的血流感染日益增加,其中念珠菌属引起的感染占真菌感染的90.0%以上,主要包括白色念珠菌(66.0%)、光滑念珠菌(11.2%)、热带念珠菌(7.6%)、近平滑念珠菌(5.6%)和克柔念珠菌(2.4%) 5种念珠菌,占临床念珠菌属感染的90.0%以上。目前,检测和鉴定念珠菌属/种血流感染主要依赖血培养和血清学试验,但固有的方法学缺陷难以满足临床快速、准确鉴定血流感染的需要。

目的:分别建立念珠菌属和5种念珠菌(白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌和克柔念珠菌)的real-time PCR快速检测平台,并对所建方法及其临床应用价值进行初步评价。

方法:

1.引物和探针设计:分别以上述5种念珠菌标准菌株的5.8S rRNA 基因(5.8S rDNA)序列和内转录间隔序列(ITS)作为参考序列,通过属、种间序列比对,在5.8S rDNA序列上设计通用引物和TaqMan-MGB探针检测念珠菌属,建立念珠菌菌属检测平台;在ITS序列上设计特异性引物和TaqMan-MGB探针检测5种念珠菌,建立念珠菌菌种检测平台。

2.方法学评价:在标准菌株制备的梯度纯菌液(纯菌液标本)、梯度模拟念珠菌感染血标本(模拟血标本)中评价所建方法的检测下限,并通过批内、批间试验评价所建方法检测精密度;在5种念珠菌临床分离株、7种细菌及人DNA中验证所建方法的准确性和特异度。

3.临床应用评价:以血培养作为金标准,联合所建念珠菌属、种检测方法同时通过对328份血标本的同步检测结果比较,初步评价所建方法临床应用价值。

4.血培养评价:将模拟血标本注入血培养瓶,通过阳性报警时间(Time to positivity , TTP)和检测下限评价血培养方法的敏感度,并与所建方法检测进行比较。

结果:

1. 引物和探针设计:(1)念珠菌菌属检测平台:通过引物和探针是否完全匹配的比对,念珠菌属平台检测5种念珠菌的理论检出率分别为白色念珠菌98.0%(599株种内比对结果),光滑念珠菌90.4%(136株),近平滑念珠菌97.9%(388株),热带念珠菌86.7%(255株)和克柔念珠菌98.0%(200株);引物序列与人、细菌、立克次氏体、病毒、衣原体、支原体DNA序列库比对检索无匹配序列。(2)念珠菌菌种检测平台:白色念珠菌的理论检出率为98.9%(468株),光滑念珠菌100%(114株),近平滑念珠菌95.5%(312株),热带念珠菌87.9%(247株)和克柔念珠菌82.5%(183株),引物序列与人、细菌、立克次氏体、病毒、衣原体、支原体序列库比对检索无匹配序列。

2. 方法学评价:(1)念珠菌菌属检测平台:纯菌液标本检测下限为101 -102 CFU/ml(白色念珠菌和克柔念珠菌的检测下限为101 CFU/ml,光滑念珠菌、近平滑念珠菌和热带念珠菌的检测下限为102 CFU/ml);模拟血标本的检测下限为101 CFU/ml(光滑念珠菌的检出率为75%,其余4种100%),批内和、批间CV值均小于5%;对5种念珠菌临床分离株的检出率为100%,对细菌、非念珠菌属真菌以及人类基因组DNA的特异度100%;328份血培养标本平行对照结果显示灵敏度和特异度分别为100%和98.4%;模拟血培养对比研究显示,优于血培养对白色念珠菌(102 CFU/ml)和光滑念珠菌(103 CFU/ml)的检测下限。

(2)念珠菌菌种检测平台:在纯菌液标本中,白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌和克柔念珠菌的检测下限皆为101 CFU/ml,批内、批间CV值皆小于5%;在模拟血标本中,上述5种念珠菌检测下限皆为101 CFU/ml;准确度评价显示,对白色念珠菌(135株)、光滑念珠菌(102株)、近平滑念珠菌(24株)、热带念珠菌(61株)和克柔念珠菌(12株)临床分离株的检出率皆为100%;对非白色念珠菌、细菌以及人类基因组DNA标本检测显示5种念珠菌检测方法的特异度皆为100%;328份平行血培养对照检测显示,5种念珠菌检测方法的准确度和特异度皆为100%。

结论:

1. 本研究成功建立了临床血流感染常见念珠菌菌属和种的real-time PCR检测平台,联合检测可将念珠菌感染直接鉴定到种水平。

2. 检测平台具有快速、准确、灵敏度高、重复性好等优势。

3. 所建检测方法适用于临床念珠菌血症的早期诊断。

英文摘要

Backgrounds: The prevalence of Candida in bloodstream infections (BSIs) has been increased. To date, the detection and identification of Candida in BSIs still mainly relies on blood culture and serological tests, but they have various limitations.

Objective: To establish real-time PCR-based assays for the detection of Candida genus and identification of Candida albicans (C. albicans), Candida glabrata (C. glabrata), Candida parapsilosis (C. parapsilosis), Candida tropicalis (C. tropicalis), and Candida krusei (C. Krusei) from whole blood samples.

Methods: The primers/probe system for detection of Candida genus was designed on the 5.8S rRNA gene (5.8S rDNA), and those for detection of Candida species were on the internal transcribed spacer (ITS). Sensitivity, specificity, reproducibility of the PCR and its clinical application were performed in the study to assess the assays.

Results:

1. The design of the primers and probes:  The Nucleotide Database in Genebank was scrutinized for the lists of known strains of the five Candida species to assess the primers and probes. (1) In the Candida genus detection assay, the forward primer showed 1 mismatch within 599 strains of C. albicans, the reverse primer showed 6 mismatches, and the probe showed 5 mismatches. So the theoretical detection rate for C. albicans was 98.0%, which was calculated as follows: (total number of strains in the database - total number of strains mismatched with the primers/probe system)/ total number of strains in the database×100%. Using the same algorithm, the theoretical rates for detecting C. glabrata, C. tropicalis, C. parapsilosis and C. krusei were 90.4%, 88.6%, 97.9% and 98.0%, respectively. (2) In the Candida species detection assays, the forward primer for detection of C. albicans showed 1 mismatch within 468 clinical strains, the reverse one showed 4 mismatches, and its probe showed 0 mismatches. So the theoretical detection rate for C. albicans was 98.9%. Using the same algorithm, the theoretical rates for detecting C. glabrata, C. tropicalis, C. parapsilosis and C. krusei were 100%, 95.5%, 87.9% and 82.5%, respectively.

2. Assay assessments: The analytic sensitivity of real-time PCR detection assay for Candida genus was 101 CFU/ml, which was superior to the sensitivity of blood culture. The analytic sensitivity of real-time PCR detection assays for Candida species were 102 CFU/ml for the five species included in the study, meanwhile, the lowest amount of Candida that the identification assays can measure accurately in suspensions was 101 CFU/ml. The analytic specificity was 100% when evaluating the assays using DNA samples from clinical isolates and human blood. The maximum CVs of intra-assay and inter-assay for the detection limit varied considerably in the different species and at different concentration, but they all met the clinical requirements and allowed unambiguous quantification at detectable magnitude orders. To assess the clinical applicability of PCR detection assay , 328 blood samples from 82 patients were prospectively tested and real-time PCR results were compared with results from blood culture. Diagnostic sensitivity of the PCR was 100% using as gold standard blood culture, and specificity was 98.4%.

Conclusion: Our data suggest that the developed assays can be used for in clinical laboratories as accurate and rapid detection and identification tests for the Candida from whole blood. Although further evaluation is warranted, our assays hold promise for earlier diagnosis of candidemia.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/125286
专题北京大学航天临床医学院
作者单位北京大学航天临床医学院
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郭毅. 血流常见真菌感染实时荧光定量PCR快速检测平台的建立及其应用研究[D]. 北京大学航天临床医学院. 北京大学,2016.
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