CN105755108A - 烟曲霉实时pcr技术 - Google Patents

烟曲霉实时pcr技术 Download PDF

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CN105755108A
CN105755108A CN201510586315.3A CN201510586315A CN105755108A CN 105755108 A CN105755108 A CN 105755108A CN 201510586315 A CN201510586315 A CN 201510586315A CN 105755108 A CN105755108 A CN 105755108A
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aspergillus fumigatus
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CN105755108B (zh
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曹国君
邢志芳
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North Hospital Of Huashan Hospital Affiliated To Fudan University
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Abstract

近20多年来，由于艾滋病患者的增多，新的治疗技术和骨髓移植、器官移植和强化化疗等广泛应用于临床，侵袭性真菌病的发病率明显升高，严重危害人类健康，甚至危及生命，大大增加了临床治疗的难度，已引起广泛高度重视，本实验室建立了一种针对烟曲霉进行特异性检测的荧光定量PCR方法(探针法)，该PCR检测体系的检测敏感性方面，线性检测范围的下限可至102copies/ml，上限可达108copies/ml，敏感性、特异性和稳定性均较好。

Description

烟曲霉实时PCR技术
本申请是分案申请，母案的申请日为2013年8月15日，申请号为201310355569.5，发明名称是烟曲霉实时PCR技术。
技术领域
近20年多年来，由于艾滋病患者的增多，新的治疗技术和骨髓移植、器官移植和强化化疗等广泛应于临床，侵入性检测设备，胃肠外营养，光谱抗生素的长期应用以及危重患者辅助机械通气代替治疗、老龄人口的增多等原因，侵袭性真菌病的发病率明显升高，特别是一些免疫功能低下患者如恶性肿瘤病人继发感染以及器官移植失败等医院内感染问题常常由真菌感染所致，严重危害人类健康，甚至危及生命，大大增加了临床治疗的难度，已引起广泛高度重视，Chandrasekar等人报道了造血干细胞移植的患者移植后出现侵袭性真菌病的患病率大于30％，而移植后因感染IFD而死亡的比率高达70％-90％，IFD的早发现早治疗对于改善患者预后具有重要意义，而当前实验室内的常规检测方法由于其敏感性和耗时性等缺陷，难以满足临床的需求，亟需寻求一种更加灵敏和特异的检测方法，为目前临床真菌感染的早期诊断、预防和治疗提供有效工具。随着近年来对真菌病原研究的不断深入，使人们重新看到了真菌PCR在临床应用的曙光，本课题组大胆求索，建立了一种针对烟曲霉病原菌进行检测的实时PCR法。
背景技术
侵袭性真菌感染(IFD)十分普遍，主要由念珠球菌属、曲霉属、隐球菌属等机会致病真菌(或条件致病真菌)引起，也可由组织胞浆菌、皮炎牙生菌、孢子丝菌等地方流行性致病真菌所致，其中曲霉菌属已成为免疫低下患者二次感染的重要致命因素，IFD的早期诊断和早期治疗对于改善病人预后有着重要意义，而当前真菌感染的特点是高院内感染率、低实验室诊断率、低临床诊断率和病情进展迅速，传统诊断方法包括深部组织培养法、组织/细胞病理学检测、1-3-β-D葡聚糖(G试验)、半乳甘露聚糖检测(GM试验)和影像学检查(如CT)等，由于它们在敏感性、特异性等方面存在缺陷，难以满足临床早期诊断的需要，如何提高IFD的早期诊断、早期治疗是国内外学者关注的热点问题，所以近年来IFD的早期诊断方法的研究被广泛开展，虽然当前仍处于实验室研究阶段，但其应用前景广阔，值得期待。
发明内容
将烟曲霉线粒体基因组大小为30696bp，将其基因组序列在http://blast.ncbi.nlm.nih.gov/Blast.cgi数据库中进行比对，找到特有的靶序列部分，在靶序列上设计一对，上游引物：5'-CTTGAATACTTCAGTAACT-3'，下游引物5'-CCTCTATTTACTAAATCATC-3'；探针5'FAM-GCAAGGCGATTTATCTCACCACT-3'TARMA，靶片段长度132bp。具体靶序列cttgaatacttcagtaactattagtaatatgttattagattctgatatatatcataatcatatagatagaagaataataatgatacaaagtggtgagataaatcgccttgctgatgatttagtaaatagagg(132bp)。
构建含靶片段的TA克隆质粒(委托宝生物公司完成)，制备梯度浓度的双份样本，具体浓度为101copies/ml、102copies/ml、103copies/ml、104copies/ml、105copies/ml、106copies/ml、107copies/ml、108copies/ml，进行PCR扩增并作融解曲线分析，验证该方法的敏感性、稳定性与特异性，同时分别以非烟曲霉菌基因组DNA，即白色念珠菌标准株(ATCC90028)基因组DNA、解脲支原体基因组DNA、人基因组DNA、人巨细胞病毒(HCMV)基因组DNA和HBV基因组DNA为模板，进一步验证该方法的特异性；将PCR产物外送检测，验证方法的可靠性。
PCR反应总体积为50μL，包括10×缓冲液(20MmMg2+plus)5μL，TaKaRaExTaqDNA聚合酶(5U/μL)0.8μL，dNTPs(各2.5mmol/L)4μL，上游引物(10μmol/L)3μL，下游引物(10μmol/L)3μL，探针(10μmol/L)3μL，DNA模板1μL，加高压灭菌后的双蒸水(ddH2O)将反应体积补足至50μL。PCR体系反应条件为：94℃预变性3min；94℃变性10s，55℃退火延伸40s，共40个循环。
PCR扩增标准曲线的斜率-3.014383，截距40.882465，相关系数R2:0.997539，提示具有较好的相关性，(说明书附图1)。该PCR检测体系的检测敏感性方面，线性检测范围的下限可至102copies/ml，上限可达108copies/ml；双份样本的检测结果总体一致(说明书附图2)，提示该PCR检测体系的重复性、稳定性较好，PCR产物外送测序，测序结果符合预期。
附图说明：图1为PCR扩增标准曲线图；图2为PCR扩增曲线。

Claims (1)

1.一种用于烟曲霉实时PCR检测的探针，其特征在于，所述探针为5'FAM-GCAAGGCGATTTATCTCACCACT-3'TARMA；所述探针的设计是针对如下靶序列：cttgaatacttcagtaactattagtaatatgttattagattctgatatatatcataatcatatagatagaagaataataatgatacaaagtggtgagataaatcgccttgctgatgatttagtaaatagagg。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102041306A (zh) * 2010-08-18 2011-05-04 李国辉 一种检测烟曲霉的dna探针、基因芯片及其应用
CN102311994A (zh) * 2011-04-19 2012-01-11 山东农业大学 烟曲霉荧光定量pcr检测方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102041306A (zh) * 2010-08-18 2011-05-04 李国辉 一种检测烟曲霉的dna探针、基因芯片及其应用
CN102311994A (zh) * 2011-04-19 2012-01-11 山东农业大学 烟曲霉荧光定量pcr检测方法

Non-Patent Citations (1)

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Title
ANA ABAD-DIAZ-DE-CERIO等: "The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR", 《MEDICAL MYCOLOGY》 *

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