CN114058695A - 泌尿道菌群检测在女性泌尿道结石诊断中的应用 - Google Patents
泌尿道菌群检测在女性泌尿道结石诊断中的应用 Download PDFInfo
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Abstract
本发明公开了泌尿道菌群检测在女性泌尿道结石诊断中的应用。所述微生物标志物包括肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)。本发明提供一种新型、无创的用于女性泌尿道结石辅助诊断的微生物标志物,检测特异性好,灵敏度高,具有指示泌尿道内结石形成的状况,可摆脱常规的影像学检查,实现泌尿道结石的简易诊断。
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,具体涉及泌尿道菌群检测在女性泌尿道结石诊断中的应用。
背景技术:
泌尿道结石(urolithiasis)泌尿系统的常见疾病,可发生于双侧肾脏、膀胱、输尿管以及尿道的任何部位。严重的泌尿道结石可以引发肾绞痛、血尿、感染、排尿困难等系列并发症,严重危害患者的健康。
迄今为止,引起泌尿道结石的病因尚未完全清晰,其中年龄、饮食、性激素水平、代谢等多种因素均被认为与泌尿道结石有关。据统计,女性罹患泌尿道结石的风险较低,但近年来随着女性的工作压力加大,不良的生活饮食习惯已导致女性泌尿道结石的患病率逐年升高。据统计,女性泌尿道结石的发病高峰分布于25~40岁及50~65岁两个年龄段。由于女性泌尿道结构的特殊性,若发病后没有得到及时的诊治,容易继发严重的严重的疼痛、感染、梗阻及肾功能不全等不良事件,因此,对结石的早期诊断、早期治疗具有重要的临床意义。
目前泌尿道结石的诊断主要依赖于泌尿道B超、X线造影、CT或MRI的影像学检查,暂无不依赖影像学的其它无创诊断手段。由于影像学仅能识别已形成一定体积的结石,因此目前临床暂无预判泌尿道结石高危人群的诊断方案,难以对具有罹患泌尿道结石风险的女性群体进行早期的预防及诊治。因此本研究开发了一套泌尿道微生态的检测方法,应用这套方案检测女性泌尿道微生物表型与泌尿道结石发病的关系,并建立应用泌尿道微生态辅助泌尿道结石的诊断、筛选泌尿道结石发病的高危人群,为泌尿道结石的精准防控提供新的手段。
发明内容
针对现有技术的匮乏,本发明的目的在于提供一种无创、不依赖影像学检查、基于泌尿道菌群含量预测女性罹患泌尿道结石风险的方案,以解决目前泌尿道结石诊断高度依赖影像学检查、诊断过程繁复、结果判别主观的临床困境,能够协助女性泌尿道泌尿道结石的疾病诊断以及指导药物研究、精准用药。
本发明发现,女性结石患者泌尿道中Lactobacillales的相对丰度明显低于无正常对照(3.76%vs 7.05%,P=0.004);而Enterobacteriales的相对丰度明显高于正常对照(3.20%vs 0.24%,P<0.001);同样的,Pseudomonadales的相对丰度明显高于正常对照(2.02%vs 0.26%,P=0.003)。经ROC分析发现,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0.747,最佳的诊断临界相对丰度为3.965%,诊断的特异性为65.5%,敏感度为85.0%;采用Enterobacteriales的相对丰度作为检验变量,ROC曲线的AUC为0.824,最佳的诊断临界相对丰度为0.350%,诊断的特异性为80.0%,敏感度为79.3%;采用Pseudomonadales的相对丰度作为检验变量,ROC曲线的AUC为0.750,最佳的诊断临界相对丰度为0.160%,诊断的特异性为70.0%,敏感度为72.4%(图3)。上述结果提示,采用上升的Enterobacteriales、Pseudomonadales以及下降的Lactobacillaless作为诊断女性泌尿道结石的新型微生物无创指标,具有较高的诊断效能。
因此,本发明的第一个目的是提供了一种无创预测女性泌尿道结石患病风险的微生物标志物,所述微生物标志物包括肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)。
本发明的第二个目的是提供了肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)作为微生物标志物在制备预测女性泌尿道结石患病风险的制剂中的应用。
本发明的第三个目的是提供检测肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂在制备预测女性泌尿道结石患病风险的制剂中的应用。
优选,所述的检测肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂是检测女性尿液中肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂。
优选,所述的尿液为连续3日清洁晨尿样品。
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。
优选地,所述的女性泌尿道结石微生物标志物相对丰度的测定方法包括宏基因组测序、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。本发明将泌尿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。
优选地,所述的试剂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。
所述的评估女性泌尿道结石发病风险的微生物标志物含量是通过检测泌尿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了提高泌尿道中Lactobacillales含量、降低泌尿道中Enterobacteriale或Pseudomonadales含量的产品在制备预防女性泌尿道结石形成的药物或功能性食品中的应用。
本发明的有益效果为:
本发明提供一种新型、无创的用于女性泌尿道结石辅助诊断的微生物标志物,检测特异性好,灵敏度高,具有指示泌尿道内结石形成的状况,可摆脱常规的影像学检查,实现泌尿道结石的简易诊断。
本发明的预测女性泌尿道结石患病风险模型灵敏度好、精确度高,只用特定的种属细菌丰度作为输入指标,不局限于测量手段,只要能获取微生物丰度值的实验手段均支持本模型方法,有利于本发明的模型实现和推广。
附图说明
图1是20例正常女性与20例女性泌尿道结石患者泌尿道微生物物种组成。
图2是女性泌尿道结石患者泌尿道特征菌群挖掘。
图3是应用特征微生物序列对女性结石患者诊断的ROC曲线分析。
具体实施方式
本发明的微生物标志物是通过新一代测序技术高通量检测女性泌尿道结石患者的泌尿道微生物组成,挖掘女性泌尿道结石患者泌尿道的微生态特征,从而确定女性泌尿道结石患者泌尿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泌尿道菌群微生态分析
(1)女性泌尿道微生物DNA提取
根据本团队早期开发的泌尿道微生态样品富集及优化的检测方案,连续三日采集清洁中段晨尿样品,每次采样50mL。所获得样品立即转运并存贮于-80℃低温冰箱,待微生物DNA提取前再进行解冻。
解冻的尿液样品采用16,000×g室温离心15分钟,弃上清后所得沉淀采用1mL生理盐水重悬洗涤,再次采用16,000×g室温离心5分钟。重复生理盐水洗涤3次后把所得沉淀采用QIAamp PowerFecal DNA Kit(Qiagen,Germany)进行尿液微生物总DNA提取。
(2)泌尿道微生物16S rRNA扩增子测序
提取后的泌尿道微生物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)进行文库定量及混合,混合文库采用1.0nmol/L的NaOH进行变性,后以14pmol/L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MiseqReporter(Illumina)进行数据拆分。
(3)泌尿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,获得泌尿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筛选女性泌尿道结石患者泌尿道特征菌群挖掘
(1)研究队列的建立
本研究收集了20例罹患泌尿道结石的女性患者清洁晨尿样品开展女性泌尿道结石患者泌尿道微生态特征分析。结石患者的入组标准为:(1)年龄≥18岁;(2)在留取尿液样本前2周内进行泌尿道超声、CT或MRI检查,以确认泌尿道结石的存在;(3)在开始留取样品前1天进行尿常规及尿培养检查,以确认无合并泌尿道感染;(4)既往无先天性泌尿道畸形;(5)排除继发性尿路结石的可能,如甲旁亢等;(6)无罹患恶性肿瘤;(6)采样前2个月内无全身或者局部的抗生素接触史。
(2)女性泌尿道结石患者泌尿道微生物检测
研究采用实施例1的方案对入组的健康志愿者进行泌尿道微生态检测,获得健康志愿者泌尿道微生物的物种组成及相对丰度列表。
(3)女性泌尿道结石患者泌尿道特征菌群的挖掘
采用线性判别分析效应大小(linear discriminant analysis effect size,LEfSe)方法分析女性泌尿道结石患者泌尿道微生态的特征菌群。首先,采用非参数检验Kruskal-Wallis秩和检验来识别健康与女性泌尿道结石的泌尿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女性泌尿道结石特征菌株的标准。分析发现,女性泌尿道中的特征菌群为升高的Enterobacteriales、Neisseriales、Pseudomonadales、Sphingomonadales、Xanthomonadales以及下降的Lactobacillales(图2)。
实施例3采用泌尿道特征微生物标记物预测女性泌尿道结石的诊断效能评估
(1)女性泌尿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49例女性泌尿道微生态样本,所有患者均经泌尿道影像学检查(B超、CT或MRI中一项或多项检查)判断有无结石情况,其中29例经影像学检查为罹患泌尿道结石,20例经影像学检查无泌尿道结石。采用实施例1的方案进行泌尿道微生态检测。
(2)采用泌尿道微生物标记物判断女性泌尿道结石的诊断效能评估
采用受试者工作曲线分析(receiver operating characteristic curve,ROC)检测应用泌尿道微生物标记物判断女性泌尿道结石的诊断效能。分析发现,采用升高的Enterobacteriales、Neisseriales、Pseudomonadales、Sphingomonadales、Xanthomonadales以及下降的Lactobacillales作为诊断模型,其中Neisseriales、Sphingomonadales及Xanthomonadales的ROC曲线计算P=0.238、P=0.067及P=0.190,提示该三个微生态指标不适合作为诊断模型的变量,因此从模型中删除。
进一步分析结果提示,女性结石患者泌尿道中Lactobacillales的相对丰度明显低于正常对照(3.76%vs 7.05%,P=0.004);而Enterobacteriales的相对丰度明显高于正常对照(3.20%vs 0.24%,P<0.001);同样的,Pseudomonadales的相对丰度明显高于正常对照(2.02%vs 0.26%,P=0.003)。经ROC分析发现,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0.747,最佳的诊断临界相对丰度为3.965%,诊断的特异性为65.5%,敏感度为85.0%;采用Enterobacteriales的相对丰度作为检验变量,ROC曲线的AUC为0.824,最佳的诊断临界相对丰度为0.350%,诊断的特异性为80.0%,敏感度为79.3%;采用Pseudomonadales的相对丰度作为检验变量,ROC曲线的AUC为0.750,最佳的诊断临界相对丰度为0.160%,诊断的特异性为70.0%,敏感度为72.4%(图3)。上述结果提示,采用上升的Enterobacteriales、Pseudomonadales以及下降的Lactobacillaless作为诊断女性泌尿道结石的新型微生物无创指标,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 (10)
1.一种无创预测女性泌尿道结石患病风险的微生物标志物,其特征在于,所述微生物标志物包括肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)。
2.肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)作为微生物标志物在制备预测女性泌尿道结石患病风险的制剂中的应用。
3.检测肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂在制备预测女性泌尿道结石患病风险的制剂中的应用。
4.根据权利要求3所述的应用,其特征在于,所述的检测肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂是检测女性尿液中肠杆菌目(Enterobacteriales)、乳杆菌目(Lactobacillales)或假单胞菌目(Pseudomonadales)相对丰度的试剂。
5.根据权利要求4所述的应用,其特征在于,所述的尿液为连续3日清洁晨尿样品。
6.根据权利要求3所述的应用,其特征在于,所述的试剂是宏基因组测序试剂。
7.根据权利要求3所述的应用,其特征在于,所述的试剂是16S rRNA扩增子测序试剂。
8.根据权利要求3所述的应用,其特征在于,所述的试剂是qPCR定量检测试剂。
9.根据权利要求3、4、5、6、7或8所述的应用,其特征在于,所述的试剂还包括聚合酶链式反应、逆转录-聚合酶链式反应或巢式PCR或核酸杂交用试剂。
10.提高泌尿道中Lactobacillales含量、降低泌尿道中Enterobacteriale或Pseudomonadales含量的产品在制备预防女性泌尿道结石形成的药物或功能性食品中的应用。
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