CN106990048B - 一种基于光声成像技术的活细菌检测方法 - Google Patents
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Abstract
一种基于光声成像技术的活细菌检测方法,涉及活细菌检测。提供可用于检测单种细菌或两种混合细菌,快速、便捷的检测益生菌与致病菌的相对浓度,为临床指导治疗提供重要参考价值的提供一种基于光声成像技术的活细菌检测方法。方法一:将光声成像增强剂加入活细菌的培养液中孵育,对探针的吞噬得细菌溶液;将所得细菌溶液取150μL,加入体积为200μL的新EP管中,进行基于光声成像技术的活细菌检测方法。方法二:将两种不同的光声成像增强剂分别加入益生菌和致病菌的培养液中进行孵育,为了保证细菌活力并促进细菌对探针的吞噬;进行基于光声成像技术的活细菌检测方法,根据所得光声强度值,计算出益生菌与致病菌的相对浓度比例。
Description
技术领域
本发明涉及活细菌检测,尤其是涉及一种基于光声成像技术的活细菌检测方法。
背景技术
微生态是人类肠道***的重要组成成分。而在人体肠道内的微生物中绝大多数是数以百兆计的存活着的细菌。这些数目庞大的细菌大致可以分为三个大类:益生菌、致病菌和中性菌。菌群的稳态,尤其是益生菌和致病菌的平衡,维系着人体的健康。益生菌主要包括:乳杆菌、酪酸梭菌、双歧杆菌、粪肠球菌、屎肠球菌、放线菌、酵母菌等,而人体的致病菌主要包括:致病性大肠埃希菌、沙门氏菌、志贺氏菌、变形杆菌、脆弱拟杆菌、耶尔森菌等。
宿主-微生物共生存在,每个人类个体的胃肠道中包含大约1013~1014个微生物体,平均约有600,000种微生物基因,不同菌属间相对丰度存在一定的个体差异。肠道菌群的多样性受遗传、饮食、年龄、环境、区域差异和抗生素使用等因素的影响,当肠道细菌的多样性下降会破坏菌群的稳定性。消化道微生态与结直肠癌及大肠腺瘤癌变密切相关。结直肠癌、腺瘤性息肉病患者与健康人群的黏膜组织或者粪便中的肠道微生物构成进行比较分析,发现微生物多样性和优势菌群降低。肠道微生物使肠道黏膜促炎症反应信号传导机制异常,导致肠道黏膜上皮损伤修复加剧,加之某些肠道微生物及其在代谢产物对肠道黏膜上皮细胞具有直接的细胞毒性作用,受损肠道黏膜上皮的不完全修复,最终导致结直肠肿瘤的形成和恶变。
随着宏基因组学、高通量测序和生物信息学分析技术的不断发展,人类对微生物群落在健康和疾病中的作用认识的越来越精确。虽然这些检测技术存在精确度高的优势,然而耗时久、成本高,无法检测活细菌的实时状态,临床实用性相对不足。因而,研发低成本、简便易操作、快速检测活细菌的方法,具有重大意义。
参考文献:
1.Nie L.and Chen X.,Structural and functional photoacoustic moleculartomography aided by emerging contrast agents.Chem Soc Rev,2014.43(20):p.7132-70。
2.Liu Y.,Nie L.and Chen X.,Photoacoustic Molecular Imaging:FromMultiscale Biomedical Applications Towards Early-Stage Theranostics.TrendsBiotechnol,2016.34(5):p.420-33。
3.Ren Y.D.,et al.,Fecal Microbiota Transplantation Induces HBeAgClearance in Patients withPositive HBeAg after Long-Term AntiviralTherapy.Hepatology,2016[Epub ahead of print]。
发明内容
本发明的目的在于提供可用于检测单种细菌或两种混合细菌,快速、便捷的检测益生菌与致病菌的相对浓度,为临床指导治疗提供重要参考价值的提供一种基于光声成像技术的活细菌检测方法。
本发明所述基于光声成像技术的活细菌检测方法之一,包括以下步骤:
1)将光声成像增强剂加入活细菌的培养液中进行孵育,为了保证细菌活力并促进细菌对探针的吞噬,得细菌溶液;
在步骤1)中,所述光声成像增强剂可选自吲哚菁绿,所述吲哚菁绿的浓度可为0.4mg/mL,吸收峰为790nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用,所述光声成像仪可采用美国Endra Nexus 128光声成像***;所述吞噬的温度可37℃,吞噬的转速可为200rpm,吞噬的时间可为8~12h。
2)将所得细菌溶液取150μL,加入体积为200μL的新EP管中,进行基于光声成像技术的活细菌检测方法。
本发明所述基于光声成像技术的活细菌检测方法之二,包括以下步骤:
1)将两种不同的光声成像增强剂分别加入益生菌和致病菌的培养液中进行孵育,为了保证细菌活力并促进细菌对探针的吞噬,益生菌为乳杆菌、酪酸梭菌、双歧杆菌、粪肠球菌、屎肠球菌、放线菌、酵母菌等中的一种;致病菌为大肠埃希菌、沙门氏菌、志贺氏菌、变形杆菌、脆弱拟杆菌、耶尔森菌等中的一种;
在步骤1)中,所述光声成像增强剂可为普鲁士蓝,所述普鲁士蓝的浓度为0.4mg/mL,吸收峰为713nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用,所述光声成像仪可采用美国Endra Nexus 128光声成像***;所述吞噬的温度可37℃,吞噬的转速可为200rpm,吞噬的时间可为8~12h。
2)将所得益生菌细菌溶液和致病菌细菌溶液各取75μL混合,加入体积为200μL的新EP管中,进行基于光声成像技术的活细菌检测方法,根据所得光声强度值,计算出益生菌与致病菌的相对浓度比例。
在步骤2)中,所述计算出益生菌与致病菌的相对浓度比例的计算公式如下:
其中,E为致病菌在不同波长处的光声信号值;F为益生菌在不同波长处的光声信号值;G为大肠杆菌与乳酸杆菌的混合菌液分别在800nm和720nm不同波长处的光声信号值,分别指示吲哚菁绿和普鲁士蓝所标记的活细菌;C为不同细菌的浓度。
本发明通过活细菌染色培养,基于光声成像技术的活细菌检测,将活细菌进行培养和染色,应用光声成像技术进行细菌检测的方法,可以用于单种活细菌的快速光声成像检测,也可用于两种活细菌的混合相对浓度鉴定。本发明的优势在于可将活菌直接用于检测,具有操作简单、检测快速、无辐射、低成本等明显优势。
附图说明
图1为基于光声成像技术的单种活细菌(乳酸杆菌)、增强剂(吲哚菁绿)及溶剂吸收峰图。在图1中,曲线a为Lac,b为Lac-ICG,c为Medium。
图2为基于光声成像技术的单种活细菌(大肠杆菌)、增强剂(普鲁士蓝)及溶剂吸收峰图。在图2中,曲线a为E.coli,b为E.coli-PB,c为Medium。
图3为基于光声成像技术的单种活细菌(乳酸杆菌)检测结果图。
图4为基于光声成像技术的单种活细菌(大肠杆菌)检测结果图。
图5为基于光声成像技术的两种活细菌(720nm波长)混合检测结果图。
图6为基于光声成像技术的两种活细菌(800nm波长)混合检测结果图。
图7为基于光声成像技术的单种活细菌检测结果图。
具体实施方式
实施例1:
参见图1~4,一种基于光声成像技术的单种活细菌检测方法,步骤如下:
(1)单种活细菌染色培养
将光声成像增强剂加入含有活细菌的培养液中进行孵育,细菌培养液依据不同菌株的生长条件选择最适培养液。为了保证细菌活力并促进细菌对探针的吞噬,采用温度37℃,转速200rmp,时间8~12h。光声增强剂一为:吲哚菁绿,其浓度为0.4mg/mL,吸收峰为790nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用。声增强剂二为:普鲁士蓝,其浓度为0.4mg/mL,吸收峰为713nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用。
(2)将上述所得细菌溶液取150μL,加入体积为200微升的新EP管中,应用美国Endra Nexus 128光声成像***进行光声成像检测。
实施例2:
参见图5~7,一种基于光声成像技术的两种活细菌混合检测方法,步骤如下:
(1)单种活细菌分别染色培养
将两种不同的光声成像增强剂分别加入含有益生菌或致病菌的培养液中进行孵育,依据不同菌株的生长条件选择最适培养液。为了保证细菌活力并促进细菌对探针的吞噬,采用温度37℃,转速200rmp,时间8~12h。光声增强剂一为:吲哚菁绿,其浓度为0.4mg/mL,吸收峰为790nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用。声增强剂二为:普鲁士蓝,其浓度为0.4mg/mL,吸收峰为713nm波长,在光声成像仪器最适波长680~980nm范围之内,且对细菌无杀伤作用。
(2)将上述所得益生菌细菌溶液和致病菌细菌溶液各取75μL混合,加入体积为200μL的新EP管中,应用美国Endra Nexus 128光声成像***进行光声成像检测。益生菌为乳杆菌、酪酸梭菌、双歧杆菌、粪肠球菌、屎肠球菌、放线菌、酵母菌等。致病菌为大肠埃希菌、沙门氏菌、志贺氏菌、变形杆菌、脆弱拟杆菌、耶尔森菌等。
(3)根据所得光声强度值,计算出益生菌与致病菌的相对浓度比例。
计算公式如下:
其中,E为致病菌在不同波长处的光声信号值;F为益生菌在不同波长处的光声信号值;G为大肠杆菌与乳酸杆菌的混合菌液分别在800nm和720nm不同波长处的光声信号值,分别指示吲哚菁绿和普鲁士蓝所标记的活细菌;C为不同细菌的浓度。
本发明旨在改进现有针对人体细菌的检测技术耗时久、成本高,无法检测活细菌的实时状态等限制。本发明的优势在于低成本、简便易操作、能够快速检测活细菌,临床实用性相对较大,具有重大意义。
Claims (8)
1.一种基于光声成像技术的活细菌检测方法,其特征在于包括以下步骤:
1)将光声成像增强剂加入活细菌的培养液中进行孵育,保证细菌活力并促进细菌对探针的吞噬,得细菌溶液;所述光声成像增强剂选自吲哚菁绿、普鲁士蓝中的一种;
2)将所得细菌溶液取150μL,加入体积为200μL的新EP管中,进行基于光声成像技术的活细菌检测方法。
2.如权利要求1所述一种基于光声成像技术的活细菌检测方法,其特征在于在步骤1)中,所述吲哚菁绿的浓度为0.4mg/mL,吸收峰为790nm波长。
3.如权利要求1所述一种基于光声成像技术的活细菌检测方法,其特征在于在步骤1)中,所述普鲁士蓝的浓度为0.4mg/mL,吸收峰为713nm波长。
4.如权利要求1所述一种基于光声成像技术的活细菌检测方法,其特征在于在步骤1)中,所述吞噬的温度为37℃,吞噬的转速为200rpm,吞噬的时间为8~12h。
5.一种基于光声成像技术的活细菌检测方法,其特征在于包括以下步骤:
1)将两种不同的光声成像增强剂吲哚菁绿和普鲁士蓝分别加入益生菌和致病菌的培养液中进行孵育,保证细菌活力并促进细菌对探针的吞噬,益生菌为乳杆菌、酪酸梭菌、双歧杆菌、粪肠球菌、屎肠球菌、放线菌、酵母菌中的一种;致病菌为大肠埃希菌、沙门氏菌、志贺氏菌、变形杆菌、脆弱拟杆菌、耶尔森菌中的一种;
2)将所得益生菌细菌溶液和致病菌细菌溶液各取75μL混合,加入体积为200μL的新EP管中,进行基于光声成像技术的活细菌检测方法,根据所得光声强度值,计算出益生菌与致病菌的相对浓度比例。
6.如权利要求5所述一种基于光声成像技术的活细菌检测方法,其特征在于所述普鲁士蓝的浓度为0.4mg/mL,吸收峰为713nm波长,在光声成像仪器波长为680~980nm,所述光声成像仪采用美国Endra Nexus 128光声成像***。
7.如权利要求5所述一种基于光声成像技术的活细菌检测方法,其特征在于在步骤1)中,所述吞噬的温度为37℃,吞噬的转速为200rpm,吞噬的时间为8~12h。
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