CN114214438B - 胆道菌群检测试剂在制备预测胆道结石术后早期复发的试剂中的应用 - Google Patents
胆道菌群检测试剂在制备预测胆道结石术后早期复发的试剂中的应用 Download PDFInfo
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Abstract
本发明公开了胆道菌群检测在预测胆道结石术后早期复发中的应用。所述的胆道菌群标志物为乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)。本发明提供一种预测胆道结石术后早期复发的微生物标志物,可用于预测胆道结石术后复发风险的辅助诊断,检测特异性好,灵敏度高,具有指示胆道微生物菌群的状况,指导胆道微生态的调整以降低胆道结石手术治疗后的疾病复发率。
Description
技术领域
本发明属于微生物技术领域以及医药技术领域,具体涉及胆道菌群检测在预测胆道结石术后早期复发中的应用。
背景技术
胆管结石(choledocholithiasis)是一种由胆道内胆汁化合物淤积形成的结石,结石形成后容易在胆管引起阻塞,引起胆汁淤滞,进而引发胆管炎、胆汁性肝硬化、门脉高压等系列并发症。据统计,全球约1.04亿人罹患胆石症,而严重的胆石症每年直接导致近10万人的死亡。由于胆管结石的具体病因尚不清晰,目前该病暂无有效的预防和内科治疗方案。手术治疗是迄今为止胆管结石患者唯一的治疗选择,但手术损伤大、术后复发率高这些治疗的局限仍然困扰着胆管结石患者。据统计,高达39%的胆管结石患者在治疗后会出现复发,需要进行二次手术。但鉴于目前临床暂无预测胆道结石患者术后复发的方案,所有胆石症患者均需要在术后进行长期的影像学监测,严重影响了其生活质量,造成庞大的经济负担。
越来越多研究证实人体微生态与机体疾病的发生、发展密切相关,且研究也发现消化道内的微生物对胆盐、胆汁酸等胆石前体成分有降解作用,因此消化道微生态检测具有预测胆石症患者疾病转归的应用前景,而其中又以胆道原位的微生物的诊断价值最大。但目前由于胆道微生物量少、消化酶多等生理特征,导致胆道微生物的研究较少。因此本研究开发了一套胆道微生态的检测方法,应用这套方案检测胆道微生物表型与胆道结石术后早期复发的关系,并建立应用胆道微生态筛选胆道结石术后早期复发高危患者的检测模型,为胆道结石手术治疗后的精准诊疗提供新的手段。
发明内容
针对现有技术的匮乏,本发明的第一个目的是提供一种预测胆道结石术后早期复发的胆道菌群标志物,所述的胆道菌群标志物为乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales),以解决目前胆道结石手术后缺乏进行早期预警、难以制定个性化精准随诊方案的临床困境,能够协助疾病诊断以及指导药物研究、精准用药。
本发明发现,4例术后复发患者胆道内Lactobacillales的相对丰度明显低于无复发患者(1.482% vs 25.040%,P= 0.040);而Fusobacteriales相对丰度明显高于于无复发患者(56.612% vs 3.466%,P= 0.001);同时,Neisseriales相对丰度亦明显高于于无复发患者(8.952% vs 0.685%,P= 0.016)。经受试者工作曲线分析(receiver operatingcharacteristic curve,ROC)发现,采用Lactobacillales的相对丰度作为检验变量,ROC曲线的曲线下面积(Area under curve,AUC)为0.814,最佳的诊断临界相对丰度为6.809%,诊断的特异性为100%,敏感度为59.0%;采用Fusobacteriales的相对丰度作为检验变量,ROC曲线的AUC为0.994,最佳的诊断临界相对丰度为23.226%,诊断的特异性为100%,敏感度为26.0%;采用Neisseriales的相对丰度作为检验变量,ROC曲线的AUC为0.821,最佳的诊断临界相对丰度为7.261%,诊断的特异性为74.0%,敏感度为75.0%(图3)。上述结果提示,采用Lactobacillale、Fusobacteriales及Neisseriales作为预测术后胆道结石复发,具有较高的诊断效能。
因此,本发明提供了预测胆道结石术后早期复发的胆道菌群标志物,所述的胆道菌群标志物为乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)。
本发明的第二个目的是提供乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)作为生物标志物在制备预测胆道结石术后早期复发的制剂中的应用。
本发明的第三个目的是提供检测乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)相对丰度的试剂在制备预测胆道结石术后早期复发的制剂中的应用。
优选,所述的检测乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)相对丰度的试剂是检测胆道胆汁中乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)相对丰度的试剂。
优选,所述的试剂是宏基因组测序试剂。
优选,所述的试剂是16S rRNA扩增子测序。
优选,所述的试剂是qPCR定量检测试剂。
即所述的检测乳杆菌目(Lactobacillale)、梭杆菌目(Fusobacteriales)或奈氏球菌目(Neisseriales)丰度是通过宏基因组测序、16S rRNA扩增子测序、微滴式数字PCR、qPCR定量检测种的任意一种或多种来测定。本发明将胆道菌群丰度或含量作为预测指标,因此,采用上述的分子生学方式均可对其进行检测,这种多样化的定量手段可打破特定实验条件和实验技能的限制,使某些不具备特定实验设备的实验室也可实验本发明的数据测量和预测。
优选,还包括聚合酶链式反应、逆转录-聚合酶链式反应、巢式PCR或核酸杂交用试剂。
所述的评估胆道结石术后早期复发风险的微生物标志物含量是通过检测胆道微生物的基因组特征性片段来确定的。所述片段为16S rRNA基因的片段。所述的扩增是通过聚合酶链反应实现的。所述的检测应用高通量测序系统实现检测。所述的微生物序列比对方案采用CLC workbench软件执行。所述的微生物序列比对采用Greengenes 13.5数据库。
本发明的第四个目的是提供提高胆道结石术后患者胆道中Lactobacillales含量、降低胆道结石术后患者胆道中的Fusobacteriales和/或Neisseriales含量的产品在制备预防胆道结石患者术后早期复发的药物中的应用。
本发明的有益效果为:
本发明提供一种预测胆道结石术后早期复发的微生物标志物,可用于预测胆道结石术后复发风险的辅助诊断,检测特异性好,灵敏度高,具有指示胆道微生物菌群的状况,指导胆道微生态的调整以降低胆道结石手术治疗后的疾病复发率。
本发明提供一种预测胆道结石术后早期复发的微生物标志物,可用于预测胆道结石术后复发风险的辅助诊断,检测特异性好,灵敏度高,具有指示胆道微生物菌群的状况,指导胆道微生态的调整以降低胆道结石手术治疗后的疾病复发率。
附图说明
图1是胆道结石患者胆道微生物物种组成;
图2是术后早期复发胆道结石患者特征微生物物种进化树;
图3是应用特征微生物序列检测结石患者术后早期复发的ROC曲线分析。
具体实施方式
本发明的微生物标志物通过新一代测序技术高通量检测胆道结石患者的胆道微生物组成,挖掘术后早期复发患者的胆道微生态特征,从而确定术后早期复发的胆道结石患者胆道菌群的特异性核苷酸序列。
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例1 胆道菌群微生态分析
(1)胆道微生物DNA提取
胆道结石的患者在内镜下乳头括约肌切开术、内镜下乳头大气囊扩张术或开腹的胆道结石清理术中,待手术医师完成了胆道结石取出术后,吸取胆汁样本3mL。所获得样品立即转运并存贮于-80°C低温冰箱,待微生物DNA提取前再进行解冻。
解冻的胆汁样品采用16,000×g室温离心5分钟,弃上清后采用1mL生理盐水重悬沉淀,再次采用16,000×g室温离心5分钟。所得沉淀采用QIAamp PowerFecal DNA Kit(Qiagen,Germany)进行胆道微生物DNA提取。
(2)胆道微生物16S rRNA扩增子测序:
提取后的胆道微生物DNA使用341F 5′-CCTACGGGNGGCWGCAG-3′及806R 5′-GGACTACHVGGGTWTCTAAT-3′作为扩增引物,UCP Multiplex PCR Kit(Qiagen)进行16S rRNA基因V3-V4区扩增,扩增后采用QIAseq Ultralow Input Library Kit进行文库构建。文库采用High Sensitivity DNA Kit(Agilent,USA)及Qubit 1× dsDNA HS Assay Kit(Thermo-Fisher,USA)进行定量及混合,混合文库采用1.0nmol/L的NaOH进行变性,后以14pM作为上机目标浓度,用HT1进行文库稀释。采用MiSeq Reagent Kit v3(Illumina,USA)测序试剂盒在Miseq平台(Illumina)上进行测序。下机数据采用Miseq Reporter(Illumina)进行数据拆分。
(3)胆道微生态扩增子数据的生物信息学分析
将Miseq Reporter拆分后的fastq数据导入CLC workbench(Qiagen)分析平台,采用Microbial Genomics Module进行扩增子数据分析。具体分析流程如下:对低质量及嵌合体数据进行过滤后,测序获得的reads比对Greengenes 13.5数据库,获得胆道微生物的组成及相对丰度列表及物种组成图(图1)。
实施例2 筛选术后早期复发的胆道结石患者的特征胆道菌群
(1)研究队列的建立
本研究收集了14例胆道结石并进行内镜下手术治疗的患者胆汁样品,术后对所有患者进行磁共振胰胆管造影(MRCP)以确定胆道结石的彻底清除。研究对所有患者在手术后进行至少12个月的随访。在12个月的随访中,患者每3个月进行一次腹部CT或者磁共振胰胆管造影以确定有无疾病复发。若患者在1年随访期间再次出现急性胆管炎症状或者影像学再次在胆道内检出结石,则患者被诊断为术后的早期复发。
(2)术后早期复发的胆道结石患者的特征胆道菌群。
胆道菌群微生态分析根据实施例1所述方法执行。根据胆道结石患者术后1年有无出现疾病复发进行分组,采用线性判别分析效应大小(linear discriminant analysiseffect size, LEfSe)方法分析术后早期复发患者胆道微生态的特征菌群。首先,采用非参数检验Kruskal-Wallis秩和检验来识别治疗后不同预后分组间胆道微生物在不同分类学水平的存在的丰度差异,然后使用线性判别分析(linear discriminant analysis,LDA)来估计每个差异丰度特征的大小。本研究采用P<0.05作为Kruskal-Wallis秩和检验中判断差异存在统计学意义的标准,采用LDA>2.0作为判断将菌群纳入早期复发特征菌株的标准。LEfSe 分析的结果通过进化树分支图进行可视化,其中研究所检测到的胆道微生物根据其进化关系建立起覆盖界、门、纲、目、科、属、种的进化分类树(图2),对于术后早期复发患者胆道增多的特征菌群,采用红色标记,对于术后早期复发患者胆道减少的特征菌群,采用绿色标记。
实施例3 采用胆道微生物标记物预测术后早期复发的胆道结石患者的诊断效能评估
(1)胆道结石患者验证队列的收集与检测
按照实施例2的方案收集43例胆道结石患者的胆汁样品,其中39例手术后1年内无发生疾病复发,4例发生疾病复发。胆道微生态检测方案按实施例1。
(2)按照实施例2的方案收集43例胆道结石患者的胆汁样品,其中39例手术后1年内无发生疾病复发,4例发生疾病复发。胆道微生态检测方案按实施例1
检测结果提示,4例术后复发患者胆道内Lactobacillales的相对丰度明显低于无复发患者(1.482% vs 25.040%,P= 0.040);而Fusobacteriales相对丰度明显高于无复发患者(56.612% vs 3.466%,P= 0.001);同时,Neisseriales相对丰度亦明显高于无复发患者(8.952% vs 0.685%,P= 0.016)。经受试者工作曲线分析(receiver operatingcharacteristic curve,ROC)发现,采用Lactobacillales的相对丰度作为检验变量,ROC曲线的曲线下面积(Area under curve,AUC)为0.814,最佳的诊断临界相对丰度为6.809%,诊断的特异性为100%,敏感度为59.0%;采用Fusobacteriales的相对丰度作为检验变量,ROC曲线的AUC为0.994,最佳的诊断临界相对丰度为23.226%,诊断的特异性为100%,敏感度为26.0%;采用Neisseriales的相对丰度作为检验变量,ROC曲线的AUC为0.821,最佳的诊断临界相对丰度为7.261%,诊断的特异性为74.0%,敏感度为75.0%(图3)。
上述结果提示,采用Lactobacillale、Fusobacteriales及Neisseriales作为预测术后胆道结石复发,具有较高的诊断效能。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.检测梭杆菌目(Fusobacteriales)相对丰度的试剂在制备预测胆道结石术后早期复发的制剂中的应用,所述的检测梭杆菌目(Fusobacteriales)相对丰度的试剂是检测胆道胆汁中梭杆菌目(Fusobacteriales)相对丰度的试剂。
2.根据权利要求1所述的应用,其特征在于,所述的试剂是qPCR定量检测试剂。
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