CN111763765B - 一种防止新冠状病毒样本rna降解的新方法 - Google Patents
一种防止新冠状病毒样本rna降解的新方法 Download PDFInfo
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Abstract
本发明涉及一种防止新冠状病毒样本RNA降解的新方法,属医疗领域。本发明创新性地将强心甙作为防止RNA降解的主要成分,实施方式有多种,为2019新型冠状病毒的检测提供了创新型思路,也可以用于防止其他种类RNA降解。
Description
技术领域
本发明涉及一种防止新冠状病毒样本RNA降解的新方法,属医疗领域。
背景技术
新型冠状病毒肺炎(Novel Coronavirus Pneumonia, NCP)是由2019新型冠状病毒(2019 Novel Coronavirus, 2019-nCoV)感染引起的一种急性传染性疾病。确定是否存在2019-nCoV感染的诊断金标准,就是能在患者体内找到2019-nCoV的存在。而要想找到2019-nCoV并明进行确判定,需要依靠测定2019-nCoV核酸。因此,保证2019-nCoV核酸检测阳性率,直接关系到疫情控制质量。
2019新型冠状病毒(2019 Novel Coronavirus, 2019-nCoV)是一种RNA型病毒。RNA病毒是病毒的一种,属于一级。它们的遗传物质是核糖核酸(RNA ribonucleic acid)。2019新型冠状病毒主要是感染呼吸道。检测和确定是否因2019新型冠状病毒感染,首先是将痰液、咽拭子样本、肺泡灌洗液等进行收集,即首先要完成标本采集、运输及保存,然后是进行荧光-PCR操作,最终确定结果。
RNA的英文名称为Ribonucleic Acid,中文名称为核糖核酸。存在于生物细胞以及部分病毒、类病毒中的遗传信息载体。RNA由核糖核苷酸经磷酸二酯键缩合而成长链状分子。因此,RNA本质上是蛋白质。RNA在离体后,很容易降解,导致检出率低甚至假阴性。导致RNA降解的原因很多,但主要原因是因为,一方面RNA极不稳定,另一方面就是样本内能够分解RNA的酶很多。由此不难看出,在样本的保存阶段,确保RNA不降解或轻微降解,是极其关键的基础性环节,直接关系到后继操作质量和检出率。因此,收集样本之后,直至进入正式检测流程之前,需要用技术手段保证样本中的病毒RNA不降解,以确保后继检测的检出率。
目前,防止样本中病毒RNA降解的方法,主要是在保存液中使用蛋白质变性剂,如异硫氰酸胍、盐酸胍、尿素、酚类等。这些常规方法,均有其不足之处。例如胍类、尿素等,必须浓度较高才可以发挥作用,由此会导致在储运过程中,温度较低时容易产生结晶和沉淀。酚类等物质本身就是一种原型质毒物,其水溶液很易通过皮肤引起全身中毒,其蒸气由呼吸道吸入,对神经***损害更大。更何况还需要加热使用,方法学方面不理想。
发明人因长期从事病理技术和诊断工作,使用的样本保存液种类较多,对样本保存技术十分熟悉。在本次新冠病毒疫情爆发后,受长期使用的含植物提取物细胞保存液技术的启发,探索出了可防止病毒样本RNA降解的新技术路线。
发明内容
发明人研究的解决方案,是要实现在病毒样本保存过程中,针对2019-nCoV核酸检测的需要来防止RNA降解。进一步地,可以针对多种病毒样本在保存中防止RNA降解。
发明人经研究发现,以支持核酸检测为目标的防止RNA降解的技术路线,要实现三个技术目标。首先是,在病毒样本进入保存液体系后,要快速对RNA进行变性,防止降解。其次是,要将样本中包含的多种可以分解RNA的酶进行失活,以防止这些酶分解RNA。最后,还要能使已经变性的RNA复活,便于进行后继扩增等检测程序。
发明人依据多年经验,经过探索实践,发现某些甙类化合物,具有快速凝固蛋白质,并且使多种酶失活的功能。甙类,也称苷类或配糖体,是一类有机化合物,其分子由一个醇基或醇样基团(配基、苷元或甙元)结合于数量不等的糖分子而构成。随即,发明人经过优选,发现甙类化合物中,强心甙比较适合于本发明的用途。强心甙(英文cardiacglycoside),又称强心配糖体,是甙类分子结构中,配基中含固醇核(甾核),其17位碳原子连以一个不饱和内酯环,其3位碳原子与糖分子相连而形成的。强心甙原本用于临床,是一类具有选择性强心作用的药物,主要用来治疗慢性心功能不全,此外又可治疗某些心律失常,尤其是室上性心律失常。但发明人发现,强心甙实际上与心肌并无特殊亲和力,而是依靠抑制Na、K-ATP酶而作用于人体的。也就是说,强心甙具有使多种酶失活的功能,但此功能并未引起病理学和检验学领域的重视。同时,发明人发现,一定浓度的强心甙溶液,可以使RNA变性,而且,在醇类的作用下,可以使RNA复活并沉淀提取。
因此,发明人通过总结和实践,创新性地将强心甙作为防止RNA降解的主要成分,将醇类(如乙醇、异丙醇等)作为复活和沉淀收集RNA的辅助试剂。
强心甙是一类甙化合物的总称,理论上都可以用于本发明,同时,强心甙的来源也很丰富,主要是被子植物的叶中,可以通过天然植物来进行提取,而且成品来源也容易解决。但每一种强心甙用于本发明的浓度不同,溶剂也不同。发明人认为一般应选用水溶性较好的品种,如毛花洋地黄苷丙。其他水溶性很差,但有机溶剂中溶解度比较好的品种,如洋地黄毒苷,则可用于特殊用途的保存液。本发明中,主要使用水溶性较好的品种,保存液也是使用水作为溶剂。
发明人在本发明的研究过程中,主要选择了毛花洋地黄苷丙。毛花洋地黄苷丙,简称毛花苷丙,是由毛花洋地黄中提取的一种速效强心苷,是去乙酰毛花苷C和地高辛的前体。发明人使用PH值为7.4的PBS缓冲液为溶剂,使用分析纯毛花苷丙,配制成浓度为0.1%—0.5%的保存液,使用分析纯异丙醇作为辅助试剂。毛花苷丙溶液优选的浓度为0.3%—0.5%,进一步优选为0.35%。使用时,将新鲜样本直接浸泡在本发明的保存液中,置于4℃冷藏存放,即可实现防止RNA降解的样本保存功能。在本发明的保存液内,RNA至少能保持72小时不降解或极少降解,对于取样、转运和到达目的地短时间保存的整个过程,时限方面已经可以满足需要。检测前,加入与保存液等量的异丙醇,混匀后,在常温下静置10-15分钟,即可沉淀提取RNA。
本发明创新使用植物提取物强心甙类物质为原料,使用安全,来源广泛,配制工艺简单,使用方便,性能良好,完全可以取代传统方法。本发明为2019新型冠状病毒的检测提供了创新型思路。
很明显,只要是使用强心甙类为原料,均在本发明的权利范围内,实施方式有多种,不限于实施例。本发明也可用于防止其他种类RNA的降解。
实施例
本发明实施过程中,所有试剂原料均为分析纯,水为双蒸水。具体实施方式如下:
1)用100g的托盘天平,称取氯化钠8.0g、氯化钾0.2g、磷酸氢二钠(十二水合)2.9g、磷酸二氢钾0.2g、毛花苷丙0.35g,备用。
2)在100ml玻璃烧杯中,加入约80ml双蒸水,加入磷酸氢二钠(十二水合)2.9g,用玻璃棒搅拌至完全溶解。再加入氯化钾和氯化钠,搅拌完全溶解。最后加入磷酸二氢钾,搅拌至完全溶解。用双蒸水加至100ml定容,配制成PBS溶液。
3)用量程为6.8-8.0的精密PH试纸,测PBS溶液的PH值为7.4左右。加入毛花苷丙,用玻璃棒搅拌至完全溶解,即为本发明的保存液。
4将本发明保存液按5ml装量分装成小瓶,贴标签即为成品。
本发明的保存液,常温保存,效期为12个月。样本加入本保存液后,需4℃冷藏保存,或使用冰袋储运。样本中的RNA在至少72小时内,不降解,或降解程度极轻微,不影响后继检测。
Claims (1)
1.一种防止新冠状病毒样本RNA降解的新方法,其特征在于,所述方法是将新鲜样本直接浸泡在保存液中,置于4℃冷藏存放;所述保存液为:使用pH值为7.4的PBS缓冲液为溶剂,使用分析纯毛花苷丙,配制而成;所述保存液中毛花苷丙的浓度为0.35g/100ml。
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