CN103725788B - 一种弓形虫tox检测试剂盒 - Google Patents
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Abstract
本发明提供一种弓形虫TOX检测试剂盒，所述试剂盒中包括核酸释放剂和PCR反应液，所述核酸释放剂包含莎梵婷（surfactin）0.01～0.5mM/L，氯化钾20～300mM/L，十二烷基磺酸钠0.01～2%和乙醇0.05～1%；所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针。使用本发明核酸释放方法与煮沸法提取核酸的检测结果无明显差异，而本发明中提取核酸时采用强烈的蛋白变性剂，快速破坏病原体外壳蛋白结构，释放出病原体核酸，无需加热即可完成DNA的释放和提取；本发明试剂盒检测TOX的灵敏度可达400copies/ml，定量线性范围为400～4.00E+09copies/ml；应用该试剂盒可以对血液和羊水等未知样本中的TOX-DNA进行快速准确的检测，为优生优育及TOX感染的早期诊断提供可靠的实验依据。
Description
技术领域
本发明提供一种弓形虫TOX检测试剂盒，具体是一种基于荧光PCR的TOX-DNA检测试剂盒。
背景技术
刚地弓形虫（Toxoplasma gondii Nicolle＆Manceaux,1908）是猫科动物的肠道球虫，由法国学者Nicolle及Manceaux在刚地梳趾鼠（Ctenodactylus gondii）的脾脏单核细胞内发现，虫体呈弓形，故命名为刚地弓形虫，简称弓形虫。
弓形虫是专性细胞内寄生虫，球虫亚纲，真球虫目，等孢子球虫科、弓形体属。该虫呈世界性分布，人和许多动物都能感染，可寄生在除红细胞外的所有有核细胞中，随血液流动，到达全身各部位，损伤大脑、心脏、眼底，致使机体免疫力下降，患各种疾病。人体感染后多处于隐性感染状态，只有当机体免疫力低下时，虫体被激活并迅速增殖，致病力增强。感染弓形虫的初孕妇女，可经胎盘血流将弓形虫传播给胎儿。在孕前3个月内感染，可造成流产、早产、畸胎或死胎，畸胎发生率高，如无脑儿、小头畸形、小眼畸形、脊柱裂等。受染胎儿或婴儿多数表现为隐性感染，有的出生后数月甚至数年才出现症状。据研究表明，婴儿出生时出现症状或发生畸形者病死率为12%，而存活者中90%有精神发育障碍，典型临床表现为脑积水、大脑钙化灶、脑膜脑炎和运动障碍；其次表现为弓形虫眼病，如视网膜脉络膜炎；此外，还可伴有发热、皮疹、呕吐、腹泻、黄疸、肝脾肿大、贫血、心肌炎、癫痫等。由于TOX对人类健康造成了很大的威胁，对这种疾病的临床感染诊断的时效性、精确性的要求也越来越高。
目前TOX感染常用的实验室诊断方法有病原学、免疫学以及分子生物学三种诊断方法。病原学诊断方法在过去近一个世纪的弓形虫病诊断中曾起到非常重要的作用，但检出率低，耗时，容易漏诊，一般不能进行生前诊断。免疫学诊断方法是目前进行弓形虫感染调查、临床弓形虫病确诊的常用方法。一般采用直接凝集试验(DAT)、间接血凝试验(IHA)、改良凝集试验(MAT)、酶联免疫吸附试验(ELISA)、补体结合试验(CT)、染色试验(DT)、间接荧光免疫试验(IFA)等十多种方法检测抗弓形虫抗体或循环抗原。其中酶联免疫吸附试验是最常用的方法，本法可检测抗弓形虫IgM、IgA、IgG、IgE抗体及Cag抗原(循环抗原)，是检测弓形虫抗体的一种可重复的方法，易自动化和标准化且敏感性强。近年来，聚核酶链式反应(PCR)法渐渐显现了其在检测上的优势，荧光PCR技术是基于传统PCR技术并结合光谱技术而发展起来的一种更灵敏，更特异，更精确的核酸检测技术。检测结果准确，重复性高，能动态反应患者治疗前、后病原体动态变化及与临床的关系，且整个过程中避免了传统PCR需后处理的问题，减少了污染。
运用PCR技术进行检测主要涉及到两个方面，核酸的提取和核酸的扩增检测。
目前国内临床上主要采用直接煮沸法对血液以及羊水样本中的弓形虫核酸进行提取，具体是向经过预处理的样本中加入某种核酸裂解液，直接煮沸，高速离心，上清即为模板；该方法核酸提取过程较复杂，样本处理耗时长，且在处理样本时经过煮沸裂解、高速离心富集DNA等多个步骤，样本中的DNA存在损耗，尤其对高浓度的样本裂解不充分、富集不完全，会造成DNA的大量丢失导致样本定量偏低，同时由于采用了水浴或金属浴的高温加热步骤，容易造成气溶胶污染。
实时荧光定量PCR技术是近年来发展迅速的一种核酸检测技术，使用一种带有荧光检测装置的PCR扩增仪，荧光检测装置能够按照一定的程序周期性地发出特定波长的激发光，收集检测荧光信号，通过检测荧光信号的动态变化实时反映PCR的每个循环的扩增水平，试验结束后可通过软件自动分析获得扩增曲线，根据扩增曲线与荧光阈值线的交点（即Ct值）以及扩增曲线的形状，可以判断阴阳性结果；如果同一反应中有已知浓度的定量参考品或标准品，则可以通过软件自动分析获得标准曲线，由此实现对未知样本的定值（即定量检测）。与传统的PCR相对比，其在反应体系中增加了一条两端分别标记荧光报告基团和淬灭基团的探针。探针结构完整时，荧光报告基团发出的荧光能量被淬灭基团吸收，呈现淬灭效应；如果扩增过程中有靶序列的存在，随着目标片段的延伸，探针分子逐渐被Taq酶水解切断，荧光报告基团与淬灭基团相互解离，阻断了二者间荧光能量转移效应，荧光报告基团发出的荧光信号被荧光检测装置收集。随着扩增的进行，荧光信号随着目的片段的扩增而呈现线性增强。试验结束后，可以通过荧光PCR仪自带的软件自动分析数据，可以获得阴阳性结果和样本浓度的定值结果，因此，该技术在靶多核苷酸样品的检测和定量分析中，已逐渐取代传统的PCR方法，得到十分广泛的应用。
目前国内外已有基于实时荧光定量PCR技术检测TOX-DNA的科研试剂盒，但这些试剂盒多半以煮沸法提取核酸，且其检测灵敏度不高，约在500～1000copies/ml左右；另外，这些试剂盒大多缺乏完善的质控体系，还需要进一步完善和提高技术水平，使此类产品更加满足临床准确诊断的需要。
发明内容
本发明提供一种核酸释放剂在弓形虫TOX检测中的应用，所述核酸释放剂包含莎梵婷（surfactin）0.01～0.5mM/L，氯化钾20～300mM/L，十二烷基磺酸钠0.01～2%和乙醇0.05～1%。
在本发明所述核酸释放剂中，其溶剂可以为本领域常用的，例如为无菌水或TE缓冲液。本发明首次披露在弓形虫TOX检测中使用含强蛋白变性剂的核酸释放剂，在很大程度上简化了该核酸检测的步骤，而且检测灵敏度有了很大的提高。
本发明提供一种弓形虫TOX检测试剂盒，所述试剂盒中包括核酸释放剂和PCR反应液，所述核酸释放剂包含莎梵婷（surfactin）0.01～0.5mM/L，氯化钾20～300mM/L，十二烷基磺酸钠0.01～2%和乙醇0.05～1%；所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针。
使用本发明试剂盒中的核酸释放剂释放核酸的方法与煮沸法提取核酸的检测结果没有明显差异，而本发明中提取核酸时采用强烈的蛋白变性剂，快速破坏病原体外壳蛋白结构，释放出病原体核酸，无需加热即可完成DNA的释放和提取；本发明试剂盒检测TOX的灵敏度可达400copies/ml，定量线性范围为400～4.00E+09copies/ml。
具体的，用于靶多核苷酸的上游引物序列为5’-CCAGACGAGACGACGCTTT-3’；下游引物序列为5’-GCATCTGGATTCCTCTCCTACC-3’；探针序列为5’-CTCCACTCTTCAATTCTCTCCGCCAT-3’。本发明的试剂盒因采用用于检测靶多核苷酸的上述引物和/或探针序列，具有很好的特异性。
本发明中，优选所述试剂盒中还包括内标，所述内标的序列为5’-CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCACGTATTCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG-3’。本发明中所述内标为插入pUC18T载体的一段长为100碱基对的人工合成DNA序列的重组体，即质粒，它作为PCR扩增体系中的阳性内对照，预防由于样本中可能存在的PCR干扰物质导致的假阴性。在所述试剂盒中包含内标的情况下，所述PCR反应液中还包括用于内标检测的上游引物、下游引物和探针；内标上游引物序列为5’-CACCACTTAAATCCTAAGGTTCCAG-3’，内标下游引物序列为5’-CTGATGACATAATTGAGATTGCACC-3’，内标探针的序列为5’-TTTTGCTGACTCACGTATTCGTAGCCAA-3’。
优选本发明所述试剂盒中还包括酶混合液，所述酶混合液中包含耐热DNA聚合酶（Taq酶）和0.5～2U/μl的尿嘧啶DNA糖基化酶（UNG酶），同时在所述PCR反应液中还包含dUTP。其中UNG酶的功能是降解含有dU的PCR产物，利用UNG酶和PCR反应液中的dUTP可以起到预防PCR产物污染的作用，从而防止样本检测假阳性。
在一个具体实施方式中，所述试剂盒中还包括TOX定量参考品、TOX阳性对照和TOX阴性对照。
本发明还提供一种TOX检测试剂盒，所述试剂盒中包括PCR反应液，所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针，且用于靶多核苷酸的上游引物序列为5’-CCAGACGAGACGACGCTTT-3’；下游引物序列为5’-GCATCTGGATTCCTCTCCTACC-3’。
本发明又提供一种TOX检测试剂盒，所述试剂盒中包括PCR反应液，所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针，且用于靶多核苷酸的探针序列为5’-CTCCACTCTTCAA TTCTCTCCGCCAT-3’。
本发明还具体提供一种TOX检测试剂盒，所述试剂盒中包括核酸释放剂、PCR反应液、内标、酶混合液、TOX定量参考品、TOX阳性对照和TOX阴性对照；且所述核酸释放剂中包含莎梵婷（surfactin）0.01～0.5mM/L，氯化钾20～300mM/L，十二烷基磺酸钠0.01～2%，乙醇0.05～1%和溶剂TE缓冲液；所述PCR反应液中包含用于靶多核苷酸扩增和检测的上游引物、下游引物和探针，用于内标扩增和检测的上游引物、下游引物和探针，10×PCR反应缓冲液，脱氧核糖核苷三磷酸和/或核糖核苷三磷酸dNTP；所述内标为插入pUC18T载体的一段长为100碱基对的人工合成DNA序列的重组体；所述酶混合液中包含DNA聚合酶和尿嘧啶DNA糖基化酶。
本发明提供的TOX荧光定量PCR检测试剂盒操作快速、方法简便、检测灵敏度高、检测范围宽，应用该试剂盒可以对血液、羊水等未知样本中的TOX-DNA进行快速准确的检测，为优生优育及诊断弓形虫感染提供可靠的实验依据。
附图说明
图1中①为实施例中TOX-DNA检测结果为阳性的待测样本的扩增曲线，图1中②为实施例中TOX-DNA检测结果为阴性的待测样本的扩增曲线。
具体实施方式
以下仅为本发明的优选实施方式，本发明的保护范围并不局限于此，任何本领域的技术人员在本发明公开的技术范围内，可很容易进行的改变或变化都涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求书的保护范围为准。
实施例1
本实施例提供一种具体的弓形虫检测试剂盒，它包括如下组分：
①核酸释放剂：包含莎梵婷（surfactin）0.1mM/L，氯化钾100mM/L，十二烷基磺酸钠（SDS）0.1%，0.1%的乙醇和溶剂TE缓冲液。
②内标（阳性内对照）：为插入pUC18T载体的一段长为100碱基对的人工合成DNA序列的重组体，即质粒，浓度为2.00E+04copies/ml，100碱基对的序列为：5’-CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCACGTATTCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG-3’。
③PCR反应液：包括10×PCR反应缓冲液5μl，0.2mmol/L的dNTP，用于靶多核苷酸扩增的上、下游引物均为0.3μmol/L，用于靶多核苷酸检测的探针为0.3μmol/L，用于内标片段扩增的上下游引物均为0.3μmol/L，用于检测内标的探针为0.1μmol/L。其中，所述10×PCR反应缓冲液为包括pH7.5的200mmol/L三羟甲基氨基甲烷盐酸盐溶液、30mmol/L氯化镁溶液、500mmol/L氯化钾溶液、0.2%的曲拉通溶液和10%的甲酰胺溶液；所述dNTP包括dATP、dCTP、dUTP和dGTP；所述用于靶多核苷酸扩增的上下游引物及用于靶多核苷酸检测的探针是源于弓形虫核酸的保守区域的引物和探针，其碱基序列分别为：上游引物：5’-CCAGACGAGACGACGCTTT-3’；下游引物：5’-GCATCTGGATTCCTCTCCTACC-3’；探针：5’-CTCCACTCTTCAATTCTCTCCGCCAT-3’；所述的用于检测内标的引物探针序列分别为：上游引物：5’-CACCACTTAAATCCTAAGGTTCCAG-3’；下游引物：5’-CTGATGACATAATTGAGATTGCACC-3’；探针：5’-TTTTGCTGACTCACGTATTCGTAGCCAA–3’。
④酶混合液：包含5U/μl的耐热DNA聚合酶（Taq酶）和1U/μl的尿嘧啶DNA糖基化酶（UNG酶）。
⑤TOX定量参考品：来源于使用TOX企业定量线性参考品L1～L5定值后的TOX强阳性质粒，该TOX定量参考品包括A、B、C、D四个浓度组成的梯度参考品，其浓度分别为4.00E+07copies/ml（A）、4.00E+06copies/ml（B）、4.00E+05copies/ml（C）、4.00E+04copies/ml（D）。
⑥TOX阳性对照：为临床医院收集的已灭活的TOX强阳性羊水样本，经浓缩离心后，加入灭菌生理盐水溶解沉淀，并经合格的TOX试剂盒检测并定值后，用灭菌生理盐水稀释至浓度为4.00E+05copies/ml。
⑦TOX阴性对照：临床医院收集的已灭活的TOX阴性羊水样本，用灭菌生理盐水稀释100倍后，经合格的TOX试剂盒检测为阴性。
实施例2
本实施例提供上述实施例1所述试剂盒用于检测血液、羊水等未知样本中TOX-DNA的操作步骤：
一、试剂准备
根据待测样本、TOX阴性对照、TOX阳性对照以及TOX定量参考品A～D的数量，按比例取相应量的PCR反应液（38μl/人份）、酶混合液（2μl/人份）及内标0.5μl/人份，充分混匀成PCR-mix，例如待测样本为3人份时，一共需配制9人份（上述四者的人份数分别为3、1、1和4）的PCR-mix；瞬时离心后备用。
二、核酸提取
1.羊水样本：取1ml样本，12,000rpm离心5分钟，弃上清后往沉淀中加入50μl核酸释放剂，与沉淀震荡或移液枪吸打混匀，作为待测样本备用。
2.血液样本：使用湖南圣湘生物科技有限公司生产的血液基因组DNA抽提试剂盒处理（该试剂盒市面有售，并不使用本发明上述实施例1中所述的核酸释放剂），得到的DNA作为待测样本。
3.TOX阴性对照、TOX阳性对照、TOX定量参考品：TOX阴性对照、TOX阳性对照、TOX定量参考品A～D分别取10μl与10μl核酸释放剂混匀待用。
三、向PCR反应管中加样
每个PCR反应管中加入上述第二步骤中处理后的待测样本、TOX阴性对照、TOX阳性对照以及TOX定量参考品A～D各10μl；静置10分钟后，每管加入步骤一中的PCR-mix40μl，吸打混匀2-3次，去除气泡后盖上管盖，2000rpm离心30秒。
四、荧光PCR反应与结果分析（在荧光定量PCR扩增仪上进行）
1）将PCR反应管放入扩增仪样品槽，按对应顺序设置待测样本名称及定量参考品浓度。
2）荧光检测通道选择：选择FAM通道（Reporter:FAM,Quencher:None）检测TOX；选择HEX或VIC通道（Reporter:VIC,Quencher:None）检测内标；参比荧光（Passive Reference）设置为none。
3）荧光定量PCR反应条件见表1：
表1
4）结果分析
反应结束后，仪器自动保存结果，可以利用仪器自带的软件进行自动分析，也可以手动调节基线的开始值、结束值以及阈值进行分析，然后记录样本Ct值和结果。扩增曲线与阈值线的交点，称为Ct值（即cycle threshold，指PCR反应管内的荧光信号达到设定的阈值时所经历的循环数值）；仪器软件根据各样本Ct值大小，通过4个浓度梯度定量参考品绘制的标准曲线，可以自动求得各样本的定量检测结果。对于测定Ct值≤39（Ct值＞0）的样本，报告为TOX-DNA阳性，此时待测样本的扩增曲线呈S型（见图1中①）；对于测定显示无Ct值的样本，同时内标检测为阳性（Ct值≤39），报告为TOX-DNA阴性，此时待测样本扩增曲线平直（见图1中②）；对于测定Ct值>39的样本，同时内标检测为阳性（Ct值≤39），报告为低于检测下限。若内标Ct值>39或内标显示无Ct值，则该样本的检测结果无效，应查找并排除原因，并对此样本进行重复试验。
使用本发明中试剂盒的特异性试验表明，其与常见病原体HBV、HCV、HIV、HCMV、RV、HSV-2、HPV等均无交叉反应，说明本发明试剂盒具有很好的特异性。
使用本发明中试剂盒检测企业工作参考品，其阴阳性符合率为100%，灵敏度检测结果符合质量标准；精密度试验表明批内和批间重复性好，其Ct值的变异系数<10%，浓度变异系数<50%。使用本发明中试剂盒对临床样本的检测试验表明，该试剂盒的定量线性范围为400～4.00E+09copies/ml，检测下限即灵敏度为400copies/ml。
Claims (5)
1.一种弓形虫TOX检测试剂盒，所述试剂盒中包括核酸释放剂和PCR反应液，所述核酸释放剂包含莎梵婷0.01～0.5mM/L，氯化钾20～300mM/L，十二烷基磺酸钠0.01～2％和乙醇0.05～1％；所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针；
用于靶多核苷酸的上游引物序列为5’-CCAGACGAGACGACGCTTT-3’；用于靶多核苷酸的下游引物序列为5’-GCATCTGGATTCCTCTCCTACC-3’；
用于靶多核苷酸的探针序列为5’-CTCCACTCTTCAATTCTCTCCGCCAT-3’。
2.根据权利要求1所述的检测试剂盒，其特征在于，所述试剂盒中还包括内标，所述内标的序列为5’-CACCACTTAAATCCTAAGGTTCCAGCTCTGTCATCCAGTTTTGCTGACTCACGTATTCGTAGCCAATCTTCTGGAGGTGCAATCTCAATTATGTCATCAG-3’。
3.根据权利要求2所述的检测试剂盒，其特征在于，所述PCR反应液中还包括用于内标检测的上游引物、下游引物和探针；且内标上游引物序列为5’-CACCACTTAAATCCTAAGGTTCCAG-3’，内标下游引物序列为5’-CTGATGACATAATTGAGATTGCACC-3’，内标探针序列为5’-TTTTGCTGACTCACGTATTCGTAGCCAA–3’。
4.根据权利要求1所述的检测试剂盒，其特征在于，所述试剂盒中还包括酶混合液，所述酶混合液中包含耐热DNA聚合酶和0.5～2U/μl的尿嘧啶DNA糖基化酶，同时在所述PCR反应液中还包含dUTP。
5.一种TOX检测试剂盒，所述试剂盒中包括PCR反应液，所述PCR反应液中包含用于靶多核苷酸扩增的上游引物、下游引物和用于靶多核苷酸检测的探针，且用于靶多核苷酸的上游引物序列为5’-CCAGACGAGACGACGCTTT-3’；下游引物序列为5’-GCATCTGGATTCCTCTCCTACC-3’；
用于靶多核苷酸的探针序列为5’-CTCCACTCTTCAATTCTCTCCGCCAT-3’。
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