CN108051494A - 一种同时检测多种遗传代谢疾病相关物质的方法和试剂盒 - Google Patents
一种同时检测多种遗传代谢疾病相关物质的方法和试剂盒 Download PDFInfo
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Abstract
本申请公开了一种同时检测多种遗传代谢疾病相关物质的方法和试剂盒。本申请方法包括(1)直接在混合内标准品中加有机溶剂或加有机溶剂和琥珀酰丙酮处理液,制成提取工作液;(2)用提取工作液孵育待测血斑样本;(3)离心,取上清液,用串联质谱检测;混合内标准品包括氨基酸、肉碱和17α羟孕酮的同位素内标准品;琥珀酰丙酮处理液中含琥珀酰丙酮内标准品和水合肼。本申请方法,一次采血就可实现四大类物质同时检测,一次性检13种氨基酸、32种肉碱、1种激素和1种酮类,能筛查46种遗传性代谢疾病,提升了新生儿筛查效率。本申请试剂盒将所有同位素内标准品加在一起使用,避免内标准品单独加入的误差和失误,保障了检测结果准确性。
Description
技术领域
本申请涉及遗传代谢疾病相关物质检测领域,特别是涉及一种同时检测多种遗传代谢疾病相关物质的方法和试剂盒。
背景技术
先天性代谢异常(inborn error of metabolism)又称为遗传代谢病(inheritedmetabolic disorders)是一类由于基因突变所导致的可对新生儿内脏、神经、肌肉、骨骼和皮肤等全身多系统组织和器官产生严重危害的代谢性疾病。在我国,每年患有出生缺陷的新生儿大约有80-120万,其中遗传代谢性疾病患儿约占50%。多年实践证明,对新生儿遗传代谢性疾病的早期筛查和早期干预能够对有效防止儿童智力低下,提高人口出生质量,且具有良好的社会和经济效益。
现有应用的新生儿代谢性疾病筛查技术有细菌抑制法、放射免疫法、荧光法等传统的遗传代谢病筛查技术,这些方法一次只能筛查一种疾病。串联质谱(MS/MS)具有超高灵敏性、高特异性、高选择性和快速检验,可以在1-2分钟内对同一个样本的多种代谢物进行同时检测,可以对氨基酸代谢障碍性疾病,脂肪酸代谢障碍性疾病,有机酸代谢障碍性疾病,激素代谢障碍性疾病等多种类型的遗传代谢病疾病进行筛查。并且,由于质谱是直接对代谢物进行检测,能够大大降低筛查假阳性和假阴性的发生率。
国际上已经有美国PerkinElmer公司开发的“非衍生化多种氨基酸、肉碱和琥珀酰丙酮测定试剂盒(串联质谱法)”;国内有广州丰华生物工程有限公司开发的“非衍生化多种氨基酸、肉碱和琥珀酰丙酮测定试剂盒(串联质谱法)”,及浙江大学开发的“氨基酸与肉毒碱串联质谱法检测试剂盒”。
无论是国际还是国内的测定试剂盒,都存在如下不足:
A、只能一次性检测部分氨基酸、肉碱和琥珀酰丙酮,而激素类的检测指标需要单独采样,单独检测;
B、新生儿筛查衍生化路线需使用酰氯,会对环境造成污染;
C、在检测氨基酸和肉碱相关的疾病、先天性肾上腺皮质增生症的时候,需要待检者两次采血,多一次采血对待测者容易造成身心影响;
D、在试验过程中,通常都是先将内标准品制成内标准品储备液,然后再由内标准品储备液配制提取工作液,在内标准品比较多的情况下,这两个步骤十分耗费实验人员的时间和精力;而且在分析物进行相对定量时,由于各个内标准品都是单独加入提取工作液中的,而每次操作都不可避免的存在操作误差,从而影响分析物浓度的准确性和检测的可重复性;并且,在众多内标准品单独加入提取工作液的过程中,还可能会出现操作失误,例如误加或少加某个或某几个内标准品,从而导致错误的检测结果。
发明内容
本申请的目的是提供一种改进的同时检测多种遗传代谢疾病相关物质的方法和试剂盒。
为了实现上述目的,本申请采用了以下技术方案:
本申请的一方面公开了一种同时检测多种遗传代谢疾病相关物质的方法,其中,多种遗传代谢疾病相关物质包括氨基酸、肉碱、酮类、激素;该方法包括以下步骤,
(1)提取工作液的制备:直接在混合内标准品中加入有机溶剂,或者加入有机溶剂和琥珀酰丙酮处理液,混合均匀,制成提取工作液;
其中,如果不检测琥珀酰丙酮,则可以不需要添加琥珀酰丙酮处理液,直接加入有机溶剂即可制成提取工作液;如果检测琥珀酰丙酮则需要加入有机溶剂和琥珀酰丙酮处理液,制成提取工作液;
(2)采用步骤(1)制备的提取工作液对待测血斑样本进行孵育;
(3)对步骤(2)孵育的产物进行离心,取上清液,采用串联质谱对上清液进行检测,获得待测血斑样本中的氨基酸、肉碱、酮类、激素信息;
其中,混合内标准品包括氨基酸同位素内标准品、肉碱同位素内标准品和17α羟孕酮同位素内标准品;琥珀酰丙酮处理液中含有琥珀酰丙酮内标准品和水合肼。
需要说明的是,本申请的检测方法,通过对提取工作液进行改进,具体的,在提取工作液中添加氨基酸同位素内标准品、肉碱同位素内标准品、17α羟孕酮同位素内标准品、琥珀酰丙酮内标准品,并采用串联质谱,可以实现氨基酸、肉碱、酮类、激素四大类物质的同时检测。本申请的方法,只需要一次采血就可以完成氨基酸代谢障碍性疾病、肉碱代谢障碍性疾病和激素代谢障碍性疾病等多种类的多项疾病的筛查,一方面缩减了检测工作步骤和时间,提高了检测效率,另一方面,也避免了多次采血对待测人员造成的痛苦或心理阴影。本申请的一种实现方式中,通过串联质谱检测,在检测干血斑样本时,仅需约3微升血液样本就可以一次性定量检测13种氨基酸、32种肉碱、1种激素和1种酮类,根据这些指标能筛查46种遗传性代谢疾病,大大提升了对新生儿遗传代谢性疾病的筛查效率。
还需要说明的是,本申请的提取工作液中,氨基酸同位素内标准品、肉碱同位素内标准品、17α羟孕酮同位素内标准品、琥珀酰丙酮内标准品可以采用市售的各种同位素标准品,例如美国剑桥实验室提供的氨基酸、肉碱、琥珀酰丙酮同位素标准品及17α羟孕酮同位素标准品,在此不做具体限定。在本申请的优选方案中,对氨基酸同位素内标准品和肉碱同位素内标准品进行了限定,详见以下方案。
优选的,步骤(2)中,孵育的条件为,30℃、700~750rpm条件下振荡30分钟。
优选的,氨基酸同位素内标准品包括L-d4-丙氨酸、L-d8-缬氨酸、L-d3-亮氨酸、L-d3-甲硫氨酸、L-13C6-苯丙氨酸、L-13C6-酪氨酸、L-d2-瓜氨酸、L-d2-鸟氨酸和L-d4;5-13C-精氨酸中的至少一种。
需要说明的是,本申请的一种实现方式中,采用L-d4-丙氨酸、L-d8-缬氨酸、L-d3-亮氨酸、L-d3-甲硫氨酸、L-13C6-苯丙氨酸、L-13C6-酪氨酸、L-d2-瓜氨酸、L-d2-鸟氨酸和L-d4;5-13C-精氨酸,九种氨基酸同位素内标准品,按照本申请的半定量方法,通过结构近似的内标定量分析物,可以对13种氨基酸进行检测。
优选的,肉碱同位素内标准品包括L-d9-游离肉碱、L-d3-乙酰肉碱、L-d3-丙酰肉碱、L-d3-丁酰肉碱、L-d9-异戊酰肉碱、L-d3-辛酰肉碱、L-d9-肉豆蔻酰肉碱和L-d3-棕榈酰肉碱中的至少一种。
需要说明的是,本申请的一种实现方式中,采用L-d9-游离肉碱、L-d3-乙酰肉碱、L-d3-丙酰肉碱、L-d3-丁酰肉碱、L-d9-异戊酰肉碱、L-d3-辛酰肉碱、L-d9-肉豆蔻酰肉碱和L-d3-棕榈酰肉碱,八种肉碱同位素内标准品,按照本申请的半定量方法,通过结构近似的内标定量分析物,可以对32种肉碱进行检测。
优选的,步骤(1)中,有机溶剂为甲醇。
本申请的另一面公开了一种同时检测多种遗传代谢疾病相关物质的试剂盒,其中,多种遗传代谢疾病相关物质包括氨基酸、肉碱、酮类、激素,该试剂盒用于采用串联质谱对待测血斑样本进行质谱检测,试剂盒包括独立包装的以下组分,
(A)混合同位素内标准品;
(B)琥珀酰丙酮处理液,琥珀酰丙酮处理液中含有琥珀酰丙酮内标准品和水合肼溶液;
(C)质控品,质控品为含有氨基酸和肉碱的人全血滤纸干血片;
其中,混合同位素内标准品由氨基酸同位素内标准品、肉碱同位素内标准品和17α羟孕酮同位素内标准品混合而成。
需要说明的是,本申请的试剂盒将多种同位素内标准品混合在一起,使用时,直接向其中添加甲醇或者添加甲醇和琥珀酰丙酮处理液,即可制备提取工作液,使用简单方便,而且避免了各种同位素内标准品单独添加到提取工作液时存在的误差或失误,进而保障了检测结果的准确性和可重复性。
其中,质控品用于评估仪器是否波动,即检测质控品中各个分析物浓度的波动范围,本申请的一种实现方式中,用变异系数CV表示,CV=标准偏差/均值,通过质控品的多次检测结果,分析各个分析物的CV值,当CV≤25%时,表示仪器状态良好,采集数据良好。
优选的,本申请的试剂盒中,氨基酸同位素内标准品包括L-d4-丙氨酸、L-d8-缬氨酸、L-d3-亮氨酸、L-d3-甲硫氨酸、L-13C6-苯丙氨酸、L-13C6-酪氨酸、L-d2-瓜氨酸、L-d2-鸟氨酸和L-d4;5-13C-精氨酸中的至少一种。
优选的,本申请的试剂盒中,肉碱同位素内标准品包括L-d9-游离肉碱、L-d3-乙酰肉碱、L-d3-丙酰肉碱、L-d3-丁酰肉碱、L-d9-异戊酰肉碱、L-d3-辛酰肉碱、L-d9-肉豆蔻酰肉碱和L-d3-棕榈酰肉碱中的至少一种。
优选的,本申请的试剂盒中还包括V型底96孔板、铝箔封口膜和使用说明书中的至少一种。
需要说明的是,V型底96孔板和铝箔封口膜可以选择性的加入试剂盒中,以方便使用,也可以单独另行购置,在此不做具体限定。使用说明书实际上就是按照本申请的方法和实施例中记载的内容所编写的试验操作步骤。
本申请的再一面公开了本申请的试剂盒,在制备遗传性代谢疾病检测药物或装置中的应用;其中,遗传性代谢疾病包括氨基酸代谢障碍性疾病、肉碱代谢障碍性疾病和激素代谢障碍性疾病中的至少一种。
优选的,本申请的一种实现方式中,尤其可以在检测氨基酸和肉碱相关疾病的同时,检测先天性肾上腺皮质增生症。
由于采用以上技术方案,本申请的有益效果在于:
本申请的检测方法,可以实现氨基酸、肉碱、酮类、激素四大类物质的同时检测,避免了衍生化路线对环境的污染,并且,在检测氨基酸和肉碱相关疾病的同时,可以对先天性肾上腺皮质增生症进行检测,只需要一次采血就可以对多项疾病进行检测,一方面,节省了人力成本,降低了多次采血操作可能带来的误差;另一方面,也避免了多次采血对待测人员的身心影响。本申请的方法,可以一次性定量检测13种氨基酸、32种肉碱、1种激素和1种酮类,根据这些指标能筛查46种遗传性代谢疾病,大大提升了对新生儿遗传代谢性疾病的筛查效率。本申请的试剂盒将所有同位素内标准品添加在一起作为混合同位素内标准品,避免了众多内标准品单独加入到提取工作液时的误差和失误,保障了检测结果的准确性和可重复性。
附图说明
图1是本申请实施例中瓜氨酸和瓜氨酸内标在鸟氨酸甲酰基转移酶缺乏症患儿和正常人的质谱分析图谱;
图2是本申请实施例中苯丙氨酸在苯丙酮尿症患儿和正常人的质谱分析图谱;
具体实施方式
本申请的检测方法和试剂盒,与市面上现有的新生儿遗传代谢性疾病筛查试剂盒相比,现有的试剂盒最多只能同时检测氨基酸、肉碱、酮类三大类物质,而本申请的试剂盒和方法,在本申请的试剂盒中加入了17α羟孕酮指标和琥珀酰丙酮指标,可以同时检测氨基酸、肉碱、酮类、激素四大类物质。特别是,氨基酸和肉碱相关疾病的筛查、先天性肾上腺皮质增生症的筛查,两者是单独分开进行的,需要两次采血,而本申请的试剂盒和方法只需要一次采血就可以完成以上两种疾病的筛查。本申请的一种实现方式中,可以一次性定量检测13种氨基酸、32种肉碱、1种激素和1种酮类,根据这些指标能筛查46种遗传性代谢疾病,实现传统的“一次实验检测一种疾病”向“一次实验检测多种疾病”的转变。
本申请的试剂盒和方法增加了肾上腺皮质增生症筛查检测的同时,提升了对于酪氨酸血症筛查的准确性。而且相对于现有的衍生化试剂盒,本申请的样本前处理步骤少,节省了人力成本,降低了人工操作可能带来的误差。同时,由于只需要一次采血,避免了多一次采血对待测对象,特别是对新生儿,造成痛苦或心理阴影。
此外,本申请的试剂盒,将多种同位素内标准品混合在一起作为混合同位素内标准品,使用简单方便,避免了各种同位素内标准品单独添加到提取工作液时存在的误差或失误,保障了检测结果的准确性和可重复性。
可以理解,操作失误可以通过熟练和细心的操作,尽量减少甚至完全避免;但是,操作误差是必然存在的,无论如何熟练和细心,每次加样的误差是不可避免的;在众多同位素内标准品单独加入到提取工作液的过程中,每个同位素内标准品的加入量存在误差,在进行重复试验的过程中,每个同位素内标准品的加入量与上一次试验的加入量也存在误差,同位素内标准品的数量越多,误差的累积就越大,从而影响分析物浓度的准确性和检测的可重复性;而本申请的试剂盒完全避免了各同位素内标准品单独加入的误差,保障了检测的准确性和可重复性。
下面通过具体实施例和附图对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明,不应理解为对本申请的限制。
实施例
本例的试剂盒包括独立包装的以下组分:
(A)混合同位素内标准品,5瓶,每瓶中混合同位素内标准品的重量为约0.3mg;每瓶混合同位素内标准品中,包括约0.15mg的氨基酸同位素内标准品、约0.15mg的肉碱同位素内标准品和约0.1μg的17α羟孕酮同位素内标准品;
其中,氨基酸同位素内标准品由等量的L-d4-丙氨酸、L-d8-缬氨酸(L-2H8-Val)、L-d3-亮氨酸(L-2H3-Leu)、L-d3-甲硫氨酸(L-2H3-Met)、L-13C6-苯丙氨酸(L-13C6-Phe)、L-13C6-酪氨酸(L-13C6-Tyr)、L-d2-瓜氨酸(L-2H2-Cit)、L-d2-鸟氨酸(L-2H2-Orn)和L-d4;5-13C-精氨酸(L-2H4;5-13C-Arg)组成;以上各氨基酸内标准品的浓度均为50nmol/mL;
肉碱同位素内标准品由15.2nmol/mL的L-d9-游离肉碱(L-2H9-C0)、3.8nmol/mL的L-d3-乙酰肉碱(L-2H3-C2)、0.76nmol/mL的L-d3-丙酰肉碱(L-2H3-C3)、0.76nmol/mL的L-d3-丁酰肉碱(L-2H3-C4)、0.76nmol/mL的L-d9-异戊酰肉碱(L-2H9-C5)、0.76nmol/mL的L-d3-辛酰肉碱(L-2H3-C8)、0.76nmol/mL的L-d9-肉豆蔻酰肉碱(L-2H9-C14)和1.52nmol/mL的L-d3-棕榈酰肉碱(L-2H3-C16)组成;
17α羟孕酮同位素内标准品为13C3-17αOHP;
(B)琥珀酰丙酮处理液,1瓶;琥珀酰丙酮处理液中含有琥珀酰丙酮内标准品13C5-SUAC和水合肼溶液,1瓶琥珀酰丙酮处理液2.5mL,将琥珀酰丙酮内标准品溶于2.5mL水合肼中,琥珀酰丙酮内标准品浓度为250nmol/mL;
(C)质控品,1套;质控品为含有氨基酸和肉碱的人全血滤纸干血片,1套质控品总计包括人全血滤纸干血片2张;其中,高浓度质控品和低浓度质控品各1张;
(D)V型底96孔板,20块;
(E)铝箔封口膜,20张;
(F)使用说明书,1份。
试剂盒于2-8℃保存。
使用说明书中披露了本例试剂盒的主要成份和使用方法,其中,主要成份包括以上(A)-(F)。使用方法包括试剂的配制,和同时检测氨基酸、肉碱、酮类和激素等多种遗传代谢疾病相关物质的方法,详细如下:
1.日常工作液制备
提取工作液配制:
(1)如果测试琥珀酰丙酮,提取工作液配制方法如下:
向1瓶混合同位素内标准品中加入9.75mL甲醇,溶解同位素内标准品,加入0.25mL琥珀酰丙酮处理液,制成10mL提取工作液。
如果不测试琥珀酰丙酮,提取工作液配制方法如下:
向1瓶混合同位素内标准品中加入10mL甲醇,溶解同位素内标准品,制成10mL的提取工作液。
2.打孔并加样
使用自动或手动打孔器在干血斑上将滤纸血片打孔,并将滤纸血片移入提供的V型截底的洁净微孔板的板孔内。纸盘的直径应约为3.2mm,即1/8英寸。
试验时,每个板孔只加入一张滤纸片。每个板设置一个孔添加高浓度质控品的3.2mm滤纸片,一个孔添加低浓度质控品3.2mm的滤纸片。
使用多道移液器以反向加样法给每个含有滤纸血片的板孔内添加90μL的提取工作液。用封膜封好,将挥发量降低到最小。
3.孵育
将微孔板置于孵育器或振荡器内,在30℃,750rpm条件下振荡30分钟。
4.琥珀酰丙酮反应
如果测试琥珀酰丙酮,则在步骤“3.孵育”完成后,室温静置2h,保证所萃取的琥珀酰丙酮充分衍生化,即确保干血片样本中的琥珀酰丙酮和提取工作液中的水合肼充分发生衍生化反应。
注意:如果不测试琥珀酰丙酮,可以省略该步骤。
5.样本提取并稀释
4000rpm,离心10min,从每个板块中将20μL上清液转移到96孔板内,加入180μL,40%乙腈进行稀释,使用封膜封好,将溶液挥发量降低到最小,在37℃,600rpm条件下振荡5分钟。
6.质谱上机检测
采用串联质谱对稀释的上清液进行检测。
7.定量结果报告
根据仪器设定程序自动得到各个分析物的浓度。
本例的试剂盒可以定量检测13种氨基酸、32种肉碱、1种激素和1种酮类。其中,13种氨基酸包括:丝氨酸、丙氨酸、精氨酸、瓜氨酸、亮氨酸、苏氨酸、甲硫氨酸、鸟氨酸、组氨酸、苯丙氨酸、酪氨酸、缬氨酸、脯氨酸。32种肉碱包括:C0、C2、C3、C4、C3DC+C4OH、C5、C5:1、C5DC+C6OH、C5OH+C4DC、C10、C10:1、C10:2、C6、C6DC、C8、C8:1、C8DC、C12、C12:1、C14、C14:1、C14:2、C14OH、C16、C16:1、C16:1OH、C16OH、C18、C18:1、C18:1OH、C18:2、C18OH。1种激素即17α羟孕酮,1种酮类即琥珀酰丙酮。9种氨基酸同位素内标准品与13种氨基酸的对应检测关系如表1所示,8种肉碱同位素内标准品与32种肉碱的对应检测关系如表1所示。
表1内标准品与分析物对应表
根据以上13种氨基酸、32种肉碱、1种激素和1种酮类的定量检测情况可以筛查46种遗传性代谢疾病,其中包括18种氨基酸代谢障碍性疾病:鸟氨酸氨甲酰基转移酶缺乏症(Ornithine transcarbamylase deficiency/OTC)、N-乙酰谷氨酸合成酶缺乏症(N-acetylglutamatesynthetase deficiency/NAGS)、枫糖尿病(Maple Syrup UrineDisease/MSUD)、高缬氨酸血症(VAL)、苯丙酮尿症(Phenylketonuria/PKU)、四氢生物喋呤缺乏症(Tetrahydrobiopterin deficiency/BH4D)、高苯丙氨酸血症Hyperphenylalaninemia/HPA、酪氨酸血症I型(Tyrosinemia I/TYR I)、酪氨酸血症II型(Tyrosinemia II/TYR II)、酪氨酸血症III型(TyrosinemiaⅢ/TYRⅢ)、精氨琥珀酸酶血症(ArgininosuccinicAciduria/ASA)、瓜氨酸血症I型(Citrullinemia I/CIT I)、瓜氨酸血症II型(Citrullinemia II/CIT II)、精氨酸血症(Argininemia/ARG)、同型半胱氨酸血症(Homocystinemia/HCY)、高甲硫氨酸血症(Hypermethioninemia/MET)、高鸟氨酸血症-高氨血症-同型瓜氨酸血症综合症(HyperammonemiaHyperornithinemiaHomocitrullinuriaSyndrome/HHH)、高鸟氨酸血症(Hyperornithinemia/ORN);12种有机酸血症:3-甲基巴豆酰辅酶A羧化酶缺乏症(3-Methyl crotonyl-CoA carboxylase deficiency/3MCC)、3-甲基戊烯二酸血症(3-Methyl glutaconicaciduria/3MGA)、2-甲基-3-羟丁酰辅酶A脱氢酶缺乏症(2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency/MHBD)、多发性羧化酶缺乏症(Multiple carboxylase deficiency/MCD)、β-酮硫酶缺乏症(Beta-ketothiolasedeficiency/BKT)、3-羟基-3-甲基戊二酰辅酶A裂解酶缺乏症(3-hydroxy-3-methylglutaryl-CoA lyase deficiency)、2-甲基丁酰基辅酶A脱氢酶缺乏症(2-Methylbutyryl CoA dehydrogenase deficiency/SBCADD)、异戊酸血症(Isovalericacidemia/IVA)、丙酸血症(Propionic acidemia/PA)、甲基丙二酸血症(MethylmalonicAcidemias/MMA)、戊二酸血症I型(Glutaricacidemia type I/GA I)、戊二酸血症II型(Glutaricacidemia type II/GA II);15种脂肪酸氧化缺陷疾病:肉碱转运缺乏症(Carnitine uptake defect/CUD)、肉毒碱棕榈酰基转移酶缺乏症I型(CarnitinePalmitoylTransferase Deficiency TypeI I/CPT I)、肉毒碱棕榈酰基转移酶缺乏症II型(Carnitine PalmitoylTransferase Deficiency TypeI II/CPT II)、中链酰基辅酶A脱氢酶缺乏症(Medium-Chain Acyl-CoA Dehydrogenase Deficiency/MCAD)、长链-3-羟酰基辅酶A脱氢酶缺乏症(3-Hydroxy Long Chain Acyl-CoA Dehydrogenase Deficiency/LCHAD)、丙二酰基辅酶A脱羧酶缺乏症(Malonyl-CoA Decarboxylase deficiency/MCD)、异丁酰基辅酶A脱氢酶缺乏症(Isobutyryl-CoA dehydrogenase deficiency/IBDH)、乙基丙二酸脑病变(Ethylmalonic encephalopathy/EE)、短链酰基辅酶A脱氢酶缺乏症(ShortChain Acyl-CoA Dehydronase Deficiency/SCAD)、肉碱/酰基肉碱移位酶缺陷(Carnitine/AcylcarnitineTranslocase Deficiency/CACT)、中短链羟酰基辅酶A脱氢酶缺乏症(Midium/Short Chain Hydroxy Acyl-CoA Dehydrogenase Deficiency/M/SCHAD)、极长链酰基辅酶A脱氢酶缺乏症(Very Long Chain Acyl-CoA DehydrogenaseDeficiency/VLCAD)、中链-3-酮酰辅酶A硫解酶缺乏症(Medium-chain ketoacyl-CoAthiolase deficiency)、2,4-二烯酰辅酶A还原酶缺乏症(2,4-Dienoyl-CoA reductasedeficiency)、三功能蛋白缺陷病(Trifunctional Protein Deficiency/TFP);以及一种激素代谢相关的疾病,即先天性肾上腺皮质增生症。
试验例:
本例对1000例血液样本进行了检测,所有血液样本由深圳华大生命科学研究院提供和保存,500例血液样本收集自全国范围内的新生儿。
按照本例提供的试剂盒的使用方法,本例首先对质控品进行了检测,以评估仪器是否波动,即检测质控品中各个分析物浓度的波动范围,用变异系数CV表示,CV=标准偏差/均值,当CV≤25%时则表示仪器状态良好,采集数据良好。本例在一天内对质控品进行了十次检测,每次两个质控品重复,一共20个试验,计算各分析物浓度的CV值,如表2所示。
表2质控品各分析物的CV值
表2的结果显示,各分析物的CV值都小于25%,证明仪器状态良好,采集数据良好,可以用于检测。
按照本例提供的试剂盒的使用方法,对1000例血液样本的血斑进行检测,结果显示,其中,筛选出鸟氨酸甲酰基转移酶缺乏症1例和苯丙酮尿症1例,结果与预期相符。鸟氨酸甲酰基转移酶缺乏症患儿及正常人质谱分析图谱见图1,苯丙酮尿症患儿及正常人质谱分析图谱见图2。
在图1中,A图和B图分别代表鸟氨酸甲酰基转移酶缺乏症患儿的瓜氨酸及瓜氨酸内标的峰形和强度,C图和D图分别代表正常人的瓜氨酸及瓜氨酸内标的峰形和强度,与他们的浓度成正比。通过比较,可以看见,鸟氨酸甲酰基转移酶缺乏症患儿的瓜氨酸强度比正常人的强度低很多,超出了正常范围。在图2中,A图和B图分别代表苯丙酮尿症患儿的苯丙氨酸及苯丙氨酸内标的峰形和强度,C图和D图分别代表正常人的苯丙氨酸及苯丙氨酸内标的峰形和强度,与他们的浓度成正比。通过比较,可以看见,苯丙酮尿症患儿的苯丙氨酸强度比正常人的强度高很多,超出了正常范围。
以上内容是结合具体的实施方式对本申请所作的进一步详细说明,不能认定本申请的具体实施只局限于这些说明。对于本申请所属技术领域的普通技术人员来说,在不脱离本申请构思的前提下,还可以做出若干简单推演或替换。
Claims (10)
1.一种同时检测多种遗传代谢疾病相关物质的方法,其特征在于:所述多种遗传代谢疾病相关物质包括氨基酸、肉碱、酮类、激素;
所述方法包括以下步骤,
(1)提取工作液的制备:直接在混合内标准品中加入有机溶剂,或者加入有机溶剂和琥珀酰丙酮处理液,混合均匀,制成提取工作液;
(2)采用步骤(1)制备的提取工作液对待测血斑样本进行孵育;
(3)对步骤(2)孵育的产物进行离心,取上清液,采用串联质谱对所述上清液进行检测,获得待测血斑样本中的氨基酸、肉碱、酮类、激素信息;
所述混合内标准品包括氨基酸同位素内标准品、肉碱同位素内标准品和17α羟孕酮同位素内标准品;
所述琥珀酰丙酮处理液中含有琥珀酰丙酮内标准品和水合肼。
2.根据权利要求1所述的方法,其特征在于:所述步骤(2)中,孵育的条件为,30℃、700~750rpm条件下振荡30分钟。
3.根据权利要求1或2所述的方法,其特征在于:所述氨基酸同位素内标准品包括L-d4-丙氨酸、L-d8-缬氨酸、L-d3-亮氨酸、L-d3-甲硫氨酸、L-13C6-苯丙氨酸、L-13C6-酪氨酸、L-d2-瓜氨酸、L-d2-鸟氨酸和L-d4;5-13C-精氨酸中的至少一种。
4.根据权利要求1或2所述的方法,其特征在于:所述肉碱同位素内标准品包括L-d9-游离肉碱、L-d3-乙酰肉碱、L-d3-丙酰肉碱、L-d3-丁酰肉碱、L-d9-异戊酰肉碱、L-d3-辛酰肉碱、L-d9-肉豆蔻酰肉碱和L-d3-棕榈酰肉碱中的至少一种。
5.根据权利要求1或2所述的方法,其特征在于:所述步骤(1)中,有机溶剂为甲醇。
6.一种同时检测多种遗传代谢疾病相关物质的试剂盒,所述多种遗传代谢疾病相关物质包括氨基酸、肉碱、酮类、激素,所述试剂盒用于采用串联质谱对待测血斑样本进行质谱检测,其特征在于:
所述试剂盒中包括独立包装的以下组分,
(A)混合同位素内标准品;
(B)琥珀酰丙酮处理液,所述琥珀酰丙酮处理液中含有琥珀酰丙酮内标准品和水合肼溶液;
(C)质控品,所述质控品为含有氨基酸和肉碱的人全血滤纸干血片;
所述混合同位素内标准品由氨基酸同位素内标准品、肉碱同位素内标准品和17α羟孕酮同位素内标准品混合而成。
7.根据权利要求6所述的试剂盒,其特征在于:所述氨基酸同位素内标准品包括L-d4-丙氨酸、L-d8-缬氨酸、L-d3-亮氨酸、L-d3-甲硫氨酸、L-13C6-苯丙氨酸、L-13C6-酪氨酸、L-d2-瓜氨酸、L-d2-鸟氨酸和L-d4;5-13C-精氨酸中的至少一种。
8.根据权利要求6所述的试剂盒,其特征在于:所述肉碱同位素内标准品包括L-d9-游离肉碱、L-d3-乙酰肉碱、L-d3-丙酰肉碱、L-d3-丁酰肉碱、L-d9-异戊酰肉碱、L-d3-辛酰肉碱、L-d9-肉豆蔻酰肉碱和L-d3-棕榈酰肉碱中的至少一种。
9.根据权利要求6-8任一项所述的试剂盒,其特征在于:所述试剂盒中还包括V型底96孔板、铝箔封口膜和使用说明书中的至少一种。
10.根据权利要求6-9任一项所述的试剂盒,在制备遗传性代谢疾病检测药物或装置中的应用;所述遗传性代谢疾病包括氨基酸代谢障碍性疾病、肉碱代谢障碍性疾病和激素代谢障碍性疾病中的至少一种;优选的,所述激素代谢障碍性疾病为先天性肾上腺皮质增生症。
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