CN113624964A - 一种侵袭性真菌的检测方法及检测试剂盒 - Google Patents
一种侵袭性真菌的检测方法及检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种侵袭性真菌的检测方法及检测试剂盒。所述检测方法为:步骤1,重组表达Dectin‑1分子胞外段蛋白,并对表达的Dectin‑1蛋白进行标记;步骤2,标记的Dectin‑1蛋白与样品中的β‑(1,3)‑D‑葡聚糖进行特异性结合反应;步骤3,通过测定标记的Dectin‑1蛋白量获得样品中β‑(1,3)‑D‑葡聚糖的含量,从而实现对侵袭性真菌的检测。所述检测试剂盒包括包被有Dectin‑1胞外段重组蛋白的酶标板、辣根过氧化酶标记的Dectin‑1胞外段重组蛋白、TMB底物溶液、终止液、阴性对照品。本发明利用Dectin‑1分子胞外段蛋白建立了稳定、可控的ELISA法和磁珠法检测真菌β‑(1,3)‑D‑葡聚糖的操作流程,该试剂盒可以代替G实验用于侵袭性真菌感染的诊断。
Description
技术领域
本发明属于生物医学诊断技术领域,具体涉及一种侵袭性真菌的检测方法及检测试剂盒。
背景技术
重症监护病房(ICU)患者由于严重的基础疾病和大型手术、各种导管的体内介入与留置,以及广谱抗菌药物和糖皮质激素的广泛应用等,常常导致免疫屏障受损与功能障碍,使得侵袭性真菌尤其是念珠菌感染的发病率不断上升,美国49所医院连续7年的监测资料表明,念珠菌在医院感染中居第4位,仅次于凝固酶阴性葡萄球菌、金黄色葡萄球菌和肠球菌,病死率则居首位。虽然目前新型、高效的氮唑类和棘白菌素类抗真菌药物不断问世,但是临床患者预后仍然不佳,病死率平均高达40%,是目前严重威胁人类健康的感染性疾病。侵袭性真菌感染起病隐匿、临床表现不典型,加之临床缺乏早期诊断指标,往往造成疾病的误诊误治,尸检研究发现75%的侵袭性真菌感染病例在生前漏诊。不能及时确诊、贻误治疗往往使得真菌感染发展成为脓毒症,导致临床抗真菌感染治疗失败。临床研究表明念珠菌血症患者中有32%发生多器官功能障碍综合征(multiple organ dysfunctionSyndrome,MODS)、30%发生脓毒症、10%发生脓毒症休克,真菌感染所导致的脓毒症已经成为ICU患者死亡的主要原因之一。所以,研究侵袭性真菌感染的早期诊断方法对于防控真菌性脓毒症、提高临床治疗成功率具有重要意义。
目前侵袭性真菌感染的诊断主要从宿主因素,临床标准和微生物学证据三个方面进行综合评估。宿主的危险因素主要包括器官移植、免疫抑制剂使用、先天性免疫缺陷等;临床标准主要是影像学、支气管镜等辅助检查的特征表现;微生物学证据主要包括直接或间接检测到真菌感染存在,是侵袭性真菌临床诊断的关键。真菌感染的实验室常规诊断方法主要依赖培养(血液或者无菌体液),并进一步通过MALDI-TOF质谱鉴定、生化鉴定或者形态学方法来确定真菌病原。但是由于真菌生长较细菌缓慢,临床标本一般需要培养72小时以上,检验报告周期长,此外标本的采集方法、部位和采集时机等也影响检验结果的客观性,常常延误患者的最佳治疗时机。另外一些全身性感染者很难承受侵入性的取材方法(如支气管肺泡灌洗液、肺组织活检、脑脊液) 等,也给真菌检验带来一定难度。所以,传统的以培养为基础的诊断方法不能满足临床早期诊断的迫切需求,临床上急需侵袭性真菌感染的早期诊断方法。
血清免疫学和分子生物学等非培养法诊断侵袭性真菌感染简便、快捷,可以弥补真菌培养报告周期长的缺陷。G实验主要检测真菌细胞壁的β-( 1,3) -D-葡聚糖,临床用于念珠菌感染的诊断。G实验在不同人群中诊断侵袭性真菌感染其敏感性和特异性有很大差异,而且检测受多种因素的影响,假阳性率高。革兰阳性菌引起的菌血症、血液透析、丙种球蛋白和白蛋白静脉输注均可以造成G实验检测结果的假阳性,而高胆红素血症和高甘油三酯血症则可造成G实验结果的假阴性。G实验基于β-( 1,3) -D-葡聚糖可与鲎血细胞提取物中的G因子α亚基结合域结合的原理检测血清中真菌β-( 1,3) -D-葡聚糖(乳胶凝集试验)。鲎是海底野生动物,目前医院检验科室所用G实验试剂均由鲎血中提取,这种利用鲎血细胞提取物的检测方法虽然能够较高灵敏地测定血中微量β-(1,3)-D-葡聚糖的含量,但是由于鲎存在不同的种系,提取物中一些杂质成分也影响检测结果,导致该方法存在一定的假阳性;另外有些地区鲎已经被列为二级保护动物,因此存在资源枯竭的可能性。
发明内容
本发明的目的是,提供一种侵袭性真菌的检测方法及检测试剂盒,主要解决现有技术中侵袭性真菌检测假阴性和假阳性较高的问题。
本发明为解决上述技术问题所采用的技术方案如下:
一种侵袭性真菌的检测方法,包括如下步骤,
步骤1,重组表达Dectin-1分子胞外段蛋白,并对表达的Dectin-1蛋白进行标记;
步骤2,标记的Dectin-1蛋白与样品中的β-(1,3)-D-葡聚糖进行特异性结合反应;
步骤3,通过测定标记的Dectin-1蛋白量获得样品中β-(1,3)-D-葡聚糖的含量,从而实现对侵袭性真菌的检测。
作为优选实施方案,所述重组表达的Dectin-1分子胞外段蛋白的氨基酸序列如SEQ ID NO: 1所示。
作为优选实施方案,重组表达Dectin-1分子胞外段蛋白的方法为:将表达Dectin-1胞外段蛋白氨基酸序列的碱基融合6×His标签并亚克隆至表达载体PCDNA3.4;利用PEI转染Expi293细胞进行重组表达,收集细胞上清液,并利用Ni-NTA磁珠进行纯化,收集纯化的蛋白即得到Dectin-1胞外段蛋白。
作为优选实施方案,采用辣根过氧化酶或生物素对Dectin-1蛋白进行标记。
作为优选实施方案,采用ELISA法或磁珠法测定样品中β-(1,3)-D-葡聚糖的含量。
作为优选实施方案,采用ELISA法测定样品中β-(1,3)-D-葡聚糖含量的方法包括如下步骤,
步骤1,将待测样品、阴性对照品加入到包被有Dectin-1胞外段重组蛋白的酶标板孔中,孵育,洗板;
步骤2,加入用辣根过氧化酶标记的Dectin-1胞外段重组蛋白,孵育,洗板;
步骤3,加入TMB底物溶液,避光反应10-20min;
步骤4,加入终止液,于酶标仪450nm波长处读吸光度值;
步骤5,以阴性孔为阴性对照,测定波长为450nm时吸光度记作N;计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
作为优选实施方案,采用磁珠法测定样品中β-(1,3)-D-葡聚糖含量的方法包括如下步骤,
步骤1,将待测样品加入到无菌96孔板内,并且加入生物素标记的Dectin-1蛋白;
步骤2,将待测样本、阴性对照品加入到96孔板孔中,孵育;
步骤3,加入链和亲霉素磁珠,震荡,充分混匀,使用磁力架进行磁珠分离;
步骤4,加入吖啶磺酰胺标记的Dectin-1-Fc融合蛋白,每孔加入化学发光液,混匀后使用多功能酶标仪,在500nm处读OD值;
步骤5,计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
作为优选实施方案,所述Dectin-1-Fc融合蛋白的氨基酸序列如SEQ ID NO: 2所示。
本发明还提供一种侵袭性真菌的检测试剂盒,包括包被有Dectin-1胞外段重组蛋白的酶标板、辣根过氧化酶标记的Dectin-1胞外段重组蛋白、TMB底物溶液、终止液、阴性对照品。
本发明还提供另一种侵袭性真菌的检测试剂盒,包括生物素标记的Dectin-1蛋白、吖啶磺酰胺标记的Dectin-1-Fc融合蛋白、链和亲霉素磁珠、化学发光液、阴性对照品。
与现有技术相比,本发明的有益效果如下:
本发明公开一种可以取代鲎G因子检测血中微量β-(1,3)-D-葡聚糖含量的检测方法。通过重组表达Dectin-1分子胞外段蛋白,建立了稳定、可控的ELISA法和磁珠法检测真菌β-(1,3)-D-葡聚糖的操作流程,该试剂盒可以代替G实验用于侵袭性真菌感染的诊断。
附图说明
图1是本发明Dectin-1蛋白磁珠法检测血液中β-(1,3)-D-葡聚糖的示意图。
具体实施方式
下面结合实施例对本发明的技术方案进行详细说明。以下采用的试剂和生物材料如未特别说明,均为商业化产品。
实施例1:Dectin-1胞外段蛋白重组表达
将表达Dectin-1胞外段蛋白(ID:Q9BXN2,65-247aa)氨基酸序列的碱基融合6×His标签并亚克隆至表达载体PCDNA3.4。利用PEI转染Expi293细胞进行重组表达,7天后,收集细胞上清液,并利用Ni-NTA磁珠进行纯化,收集到纯度达到95%以上的蛋白。重组表达的Dectin-1胞外段蛋白氨基酸序列如SEQ ID NO: 1所示。
实施例2:Dectin-1蛋白胞外段与人IgG1 Fc融合蛋白(Dectin-1-Fc)重组表达
将测序分析所获得的抗原结合蛋白A3、G1和F6的可变区编码序列分别融合人免疫球蛋白γ1(IgG1)并亚克隆至表达载体PCDNA3.4。利用PEI转染Expi293细胞进行重组表达,7天后,收集细胞上清液,并利用ProteinA磁珠进行纯化。收集到纯度达到95%以上的蛋白,得到Dectin-1-Fc蛋白,氨基酸序列如SEQ ID NO: 2所示。
实施例3:生物素(Biotin)标记Dectin-1胞外段蛋白
(1)将实施例1中制备的Dectin-1胞外段蛋白使用1×PBS缓冲液,调整浓度至浓度1mg/mL;
(2)称取NHS-biotin(生工:C100212,分子量341.4),使用 DMSO溶解,配制10mM的NHS-biotin;(避光、称取生物素时使瓶子降至室温再打开称量);
(3)按照Dectin-1胞外段蛋白:NHS-biotin=1:20摩尔比,混合后装入棕色样品管中,180rpm,室温偶联30min;
(4)使用PBS缓冲液在小置换管(10kd)中进行置换,去除未标记的Biotin和DMSO,10000g离心10min,置换三次,使DMSO含量小于1%;
(5)将标记好的抗原蛋白避光保存于-80℃,备用。
实施例4:使用吖啶酯标记Dectin-1-Fc融合蛋白
(1)将Dectin-1-Fc融合蛋白使用1×PBS缓冲液,调整浓度为1mg/mL;
(2)称取吖啶酯磺酰胺NSP-SA-NHS,使用DMF溶解,配制1 mg/ml的NSP-SA-NHS母液;
(3)向Dectin-1-Fc融合蛋白样品中加入稀释的NSP-SA-NHS溶液(抗体:NSP-SA-NHS=1:20摩尔比);
(4)将样品装入棕色样品管中,摇床180rpm,室温偶联120min;
(5)使用PBS缓冲液在小置换管(50kd)中进行置换,去除未标记的NSP-SA-NHS和DMF,10000g离心10min,置换三次,使DMF含量小于1%;
(6)将标记好的蛋白保存于-80°C,备用。
实施例5:利用Dectin-1蛋白ELISA法检测血液中β-(1,3)-D-葡聚糖
1. 取300μl待测血清加入到离心管中,加入100μl样品处理液(0.01mol/L EDTA溶液);
2. 涡旋振荡10秒,将离心管放入水浴锅内100℃加热3 min;
3. 将离心管从水浴锅中取出,小心放入离心机内,4℃,10,000g离心10 min,取上清;
4. 将包被有Dectin-1胞外段重组蛋白的酶标板取出,置于室温30min;
5. 将待测样本100μl、阴性对照品加入到酶标板孔中,37℃孵育30min;使用洗板机PBST溶液洗板3次;
6. 加入用辣根过氧化酶标记的Dectin-1胞外段重组蛋白(使用商品化的试剂盒进行标记)100μl;37℃孵育30min后,使用洗板机PBST溶液洗板3次;
7. 加入TMB底物溶液100μl,37℃避光反应15min;
8. 每孔加入50μl终止液(2M硫酸溶液),于酶标仪450nm波长处读吸光度值;
9. 以阴性孔为阴性对照,测定波长为450nm时吸光度记作N。计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
按照上述ELISA法检测方式,检测10例侵袭性真菌(白念珠菌)感染患者血清中β-(1,3)-D-葡聚糖,使用健康人血清作为阴性对照,结果如表1。
表1
样品编号 | OD值 | P/N |
1 | 1.63 | 2.76 |
2 | 2.62 | 4.44 |
3 | 3.69 | 6.25 |
4 | 2.17 | 3.68 |
5 | 2.67 | 4.53 |
6 | 1.38 | 2.34 |
7 | 2.56 | 4.34 |
8 | 3.06 | 5.19 |
9 | 1.69 | 2.86 |
10 | 2.62 | 4.44 |
阴性对照 | 0.59 |
由表1数据结果可以看出:10例真菌样本均检测出为阳性,与实际相符。
实施例6:利用Dectin-1蛋白磁珠法检测血液中β-(1,3)-D-葡聚糖
如图1所示,为Dectin-1蛋白磁珠法检测血液中β-(1,3)-D-葡聚糖的示意图。
1. 取300μl待测血清加入到离心管中,加入100μl样品处理液(0.01mol/L EDTA溶液);
2. 涡旋振荡10秒,将离心管放入水浴锅内100℃加热3 min;
3. 将离心管从水浴锅中取出,小心放入离心机内,4℃,10,000g离心10 min;
4. 将待测样品加入到无菌96孔板内,并且加入生物素标记的Dectin-1蛋白(1μg/孔);
5. 将待测样本100μl、阴性对照品加入到96孔板孔中,37℃孵育30min;
6. 加入100μl链和亲霉素磁珠,震荡15S,充分混匀,使用磁力架进行磁珠分离;
7. 加入吖啶磺酰胺标记的Dectin-1-Fc融合蛋白(1μg/孔),每孔加入化学发光液200 μl(发光液1:0.1mol/L HNO3溶解1% H2O2;发光液2:0.1mol/L NaOH;发光液1和发光液2按照1:1混合),混匀后使用多功能酶标仪,在500nm处读OD值。计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
按照上述磁珠法检测方式,检测10例侵袭性真菌(白念珠菌)感染患者血清中β-(1,3)-D-葡聚糖,使用健康人血清作为阴性对照,结果如表2。
表2
样品编号 | OD值 | P/N |
1 | 2.13 | 4.63 |
2 | 3.65 | 7.93 |
3 | 3.17 | 6.89 |
4 | 2.89 | 6.28 |
5 | 3.09 | 6.72 |
6 | 2.13 | 4.63 |
7 | 3.02 | 6.57 |
8 | 2.95 | 6.41 |
9 | 1.96 | 4.26 |
10 | 2.38 | 5.17 |
阴性对照 | 0.46 |
由表2数据结果可以看出:10例真菌样本均检测出为阳性,与实际相符。
上述仅为本发明的部分优选实施例,本发明并不仅限于实施例的内容。对于本领域中的技术人员来说,在本发明技术方案的构思范围内可以有各种变化和更改,所作的任何变化和更改,均在本发明保护范围之内。
序列表
<110> 上海市第十人民医院
<120> 一种侵袭性真菌的检测方法及检测试剂盒
<141> 2021-06-29
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 188
<212> PRT
<213> C型凝集素受体(Dectin-1)
<400> 1
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Gly Leu Leu Pro Ser Met His His His His His His
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<210> 2
<211> 414
<212> PRT
<213> 凝集素-人IgG1 Fc融合蛋白(Dectin-1-Fc)
<400> 2
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Claims (10)
1.一种侵袭性真菌的检测方法,其特征在于:该方法包括如下步骤,
步骤1,重组表达Dectin-1分子胞外段蛋白,并对表达的Dectin-1蛋白进行标记;
步骤2,标记的Dectin-1蛋白与样品中的β-(1,3)-D-葡聚糖进行特异性结合反应;
步骤3,通过测定标记的Dectin-1蛋白量获得样品中β-(1,3)-D-葡聚糖的含量,从而实现对侵袭性真菌的检测。
2.如权利要求1所述的侵袭性真菌的检测方法,其特征在于:所述重组表达的Dectin-1分子胞外段蛋白的氨基酸序列如SEQ ID NO: 1所示。
3.如权利要求1所述的侵袭性真菌的检测方法,其特征在于,重组表达Dectin-1分子胞外段蛋白的方法为:将表达Dectin-1胞外段蛋白氨基酸序列的碱基融合6×His标签并亚克隆至表达载体PCDNA3.4;利用PEI转染Expi293细胞进行重组表达,收集细胞上清液,并利用Ni-NTA磁珠进行纯化,收集纯化的蛋白即得到Dectin-1胞外段蛋白。
4.如权利要求1所述的侵袭性真菌的检测方法,其特征在于:采用辣根过氧化酶或生物素对Dectin-1蛋白进行标记。
5.如权利要求1所述的侵袭性真菌的检测方法,其特征在于:采用ELISA法或磁珠法测定样品中β-(1,3)-D-葡聚糖的含量。
6.如权利要求4所述的侵袭性真菌的检测方法,其特征在于:采用ELISA法测定样品中β-(1,3)-D-葡聚糖含量的方法包括如下步骤,
步骤1,将待测样品、阴性对照品加入到包被有Dectin-1胞外段重组蛋白的酶标板孔中,孵育,洗板;
步骤2,加入用辣根过氧化酶标记的Dectin-1胞外段重组蛋白,孵育,洗板;
步骤3,加入TMB底物溶液,避光反应10-20min;
步骤4,加入终止液,于酶标仪450nm波长处读吸光度值;
步骤5,以阴性孔为阴性对照,测定波长为450nm时吸光度记作N;计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
7.如权利要求4所述的侵袭性真菌的检测方法,其特征在于:采用磁珠法测定样品中β-(1,3)-D-葡聚糖含量的方法包括如下步骤,
步骤1,将待测样品加入到无菌96孔板内,并且加入生物素标记的Dectin-1蛋白;
步骤2,将待测样本、阴性对照品加入到96孔板孔中,孵育;
步骤3,加入链和亲霉素磁珠,震荡,充分混匀,使用磁力架进行磁珠分离;
步骤4,加入吖啶磺酰胺标记的Dectin-1-Fc融合蛋白,每孔加入化学发光液,混匀后使用多功能酶标仪,在500nm处读OD值;
步骤5,计算待测样本与阴性样本值之比P/N,当P/N>2时为阳性,当2 >P/N >1. 5时为可疑,当P/N< 1. 5时为阴性。
8.如权利要求6所述的侵袭性真菌的检测方法,其特征在于:所述Dectin-1-Fc融合蛋白的氨基酸序列如SEQ ID NO: 2所示。
9.一种侵袭性真菌的检测试剂盒,其特征在于:包括包被有Dectin-1胞外段重组蛋白的酶标板、辣根过氧化酶标记的Dectin-1胞外段重组蛋白、TMB底物溶液、终止液、阴性对照品。
10.一种侵袭性真菌的检测试剂盒,其特征在于:包括生物素标记的Dectin-1蛋白、吖啶磺酰胺标记的Dectin-1-Fc融合蛋白、链和亲霉素磁珠、化学发光液、阴性对照品。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101591667A (zh) * | 2009-05-13 | 2009-12-02 | 中国人民解放军第三军医大学第三附属医院 | Dectin-1融合蛋白表达载体及应用 |
CN101679949A (zh) * | 2007-02-23 | 2010-03-24 | 贝勒研究院 | 通过dectin-1激活人抗原呈递细胞的治疗性应用 |
US20140127267A1 (en) * | 2011-04-08 | 2014-05-08 | The General Hospital Corporation | Fungal-derived carbohydrate-conjugated scaffold |
WO2017004563A1 (en) * | 2015-07-02 | 2017-01-05 | Cidara Therapeutics, Inc. | Multi-specific binding compounds |
CN107918011A (zh) * | 2016-10-09 | 2018-04-17 | 徐新平 | 真菌的检测方法及检测试剂盒 |
CN112229994A (zh) * | 2020-12-10 | 2021-01-15 | 丹娜(天津)生物科技股份有限公司 | 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒 |
CN112940114A (zh) * | 2021-02-09 | 2021-06-11 | 丹娜(天津)生物科技股份有限公司 | 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用 |
-
2021
- 2021-06-29 CN CN202110728342.5A patent/CN113624964A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101679949A (zh) * | 2007-02-23 | 2010-03-24 | 贝勒研究院 | 通过dectin-1激活人抗原呈递细胞的治疗性应用 |
CN101591667A (zh) * | 2009-05-13 | 2009-12-02 | 中国人民解放军第三军医大学第三附属医院 | Dectin-1融合蛋白表达载体及应用 |
US20140127267A1 (en) * | 2011-04-08 | 2014-05-08 | The General Hospital Corporation | Fungal-derived carbohydrate-conjugated scaffold |
WO2017004563A1 (en) * | 2015-07-02 | 2017-01-05 | Cidara Therapeutics, Inc. | Multi-specific binding compounds |
CN107918011A (zh) * | 2016-10-09 | 2018-04-17 | 徐新平 | 真菌的检测方法及检测试剂盒 |
CN112229994A (zh) * | 2020-12-10 | 2021-01-15 | 丹娜(天津)生物科技股份有限公司 | 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒 |
CN112940114A (zh) * | 2021-02-09 | 2021-06-11 | 丹娜(天津)生物科技股份有限公司 | 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用 |
Non-Patent Citations (2)
Title |
---|
LISA M. GRAHAM 等: "Soluble Dectin-1 as a tool to detect β-glucans", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 314, pages 164 - 169 * |
孙雯雯;刘丁;王政;陈萍;王豪;成瑶;: "真菌β-葡聚糖受体Dectin-1胞外识别区的克隆和原核表达", 免疫学杂志, no. 04, pages 309 - 319 * |
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