CN112316166B - 一种生物液体样本中病原体的核黄素光化学灭活方法 - Google Patents
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Abstract
本发明属于病原体灭活技术领域,具体涉及一种生物液体样本中病原体的核黄素光化学灭活方法。针对现有的核黄素病原体灭活方法中存在的问题,本发明的技术方案是一种生物液体样本中病原体的核黄素光化学灭活方法:包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,所述光为波长范围选自360‑370nm和/或390‑400nm范围内的窄谱紫外光。本发明还进一步优选了光照时间、光照强度和核黄素浓度等参数。采用本发明的技术方案,能够达到优良的病原体灭活效果,且对生物液体样本中其他成分的损伤小。
Description
技术领域
本发明属于病原体灭活技术领域,具体涉及一种生物液体样本中病原体的核黄素光化学灭活方法。
背景技术
在医学或生物学中,常常出现生物液体样本中存在病原体需要灭活的情况。例如在血液制品中,采集、转运等过程中常常引入血传播病原体,这些血传播病原体包括病毒、细菌、原虫和螺旋体等。
随着血液病原体检测技术的发展,输血感染风险大大降低,但病原体检测窗口期依然存在、目前常规检测方法不能覆盖所有已知经血传播病原体。新发、再发经血传播病原体依旧威胁着输血安全,特别是血小板(PLT)常规储存在20-24℃条件下,细菌污染风险更高,持续稳定的血源性细菌污染成为了对血液供应机构威胁最大的因素。
发展病原体灭活技术可以有效降低输血感染风险,目前具有发展前景的血液病原体灭活技术主要有补骨脂素和核黄素,均可用于血浆、血小板和红细胞病原体灭活。补骨脂素可能存在基因毒性,因此使用后需要去除。而核黄素(维生素B2)是人体必须的天然维生素,其分解产物本身广泛存在于人体血液和组织中,并且具有天然的光化学反应,使用后不需去除,被广泛应用于血液成分病原体灭活中。
例如,中国专利申请“CN201910975223.2利用核黄素光化学法灭活血液成分病原体的设备及方法”中提出了一种核黄素光化学法灭活血液中病原体的方法,且对灭活的条件进行了优选。该专利申请中公开的较优条件为采用309nm-313nm窄谱紫外光,光照时间范围5-40min为最佳,光照能量范围0.4J/ml-3J/ml为最佳。但是,在该条件下,对病原体的灭活效果仍然不够理想,且对血液产品中的其它成分仍然具有一定的损伤。正是由于上述原因。虽然核黄素病原体灭活系统灭活血液中病原体在欧洲等国家使用,然而其在美国尚未得到FDA批准。
发明内容
针对现有的核黄素病原体灭活方法中存在的问题,本发明提供一种生物液体样本中病原体的核黄素光化学灭活方法,其目的在于:通过优化光照的波长范围,使得核黄素光化学灭活方法对病原体的灭活效果更好,且对血液成分中造成的损伤更低。
一种生物液体样本中病原体的核黄素光化学灭活方法,包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,所述光为波长范围选自360-370nm和/或390-400nm范围内的窄谱紫外光。
优选的,光为波长范围选自390-400nm范围内的窄谱紫外光。
优选的,光为波峰为395nm的紫外光。
优选的,光辐射生物液体样本的光照时间为3-30min,光照能量范围为0.2-5J/ml。
优选的,光辐射生物液体样本的光照时间为3-4.9min,光照能量范围为0.2-0.39J/ml。
优选的,核黄素在生物液体样本中的加入量为40-60μM。
优选的,核黄素在生物液体样本中的加入量为50μM。
优选的,生物液体样本为血液产品、血液产品、细胞产品或肿瘤细胞样本。
优选的,血液产品为全血、白细胞减少的全血、包装红细胞、手工血小板、单采血小板、血浆或冷沉淀。
本发明对核黄素光化学灭活生物液体样本(例如血液产品)中的病原体的方法进行改进,优选了光照波长。在相同的光照时间和光照强度的条件下,本发明优选的波长范围的光照对病原体具有更好的灭活效果。此外,由于该波长范围内的光照对病原体的灭活效果更好,因而在实际操作中,选用该波长范围内的窄谱紫外光可在更短的光照时间和更低的光照强度下对生物液体样本进行病原体灭活,从而降低光照对生物液体样本中的其他成分的损伤。
本发明的方法适用于血液产品中的病原体灭活、各种生物液体样本的去污处理、临床重度感染和肿瘤患者的治疗等,应用广泛。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1核黄素光化学灭活血液产品中的病原体的方法中采用不同波长范围的窄谱紫外光对大肠杆菌的灭活效果;
图2波长为395±5nm的窄谱紫外光和波长范围为309-313nm的窄谱紫外光对血小板中的金黄色葡萄球菌的灭活效果。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的说明。
本发明中核黄素光化学灭活生物液体样本中的病原体的方法的具体操作及装置可参考现有技术中已公开的内容进行。以下实施例中的具体操作和装置采用与中国专利申请“CN201910975223.2利用核黄素光化学法灭活血液成分病原体的设备及方法”中公开的方法和装置一致,区别在于光照波长范围、光照时间、光照能量和核黄素的浓度等进行了改变。
实施例1
本实施例选择了一系列波长范围的LED灯珠对含有50μM核黄素的健康献血者的血浆进行照射,光照时间为15min,光照强度为1W。然后通过Reed-Muench法检测血液产品中的大肠杆菌生长量。
结果如图1所示,相同光照时间和光照强度下,在一系列的窄谱紫外光中,波长为365±5nm和波长为395±5nm的LED灯珠在核黄素光化学病原体灭活系统中对大肠杆菌有较好的病原体灭活效果。
实施例2
本实施例选择了波长为395±5nm的LED灯珠和波长为309-313nm的荧光灯管对含有50μM核黄素的单采血小板进行照射,光照时间均为30min,光照强度分别为1W(395±5nm)和9W(309-313nm)。然后通过Reed-Muench法检测血液产品中的金黄色葡萄球菌的生长量。
结果如图2所示,图中还给出了未通过核黄素光化学灭活的对照实验组的数据。从图中可以看出,虽然395±5nm的LED灯珠和波长为309-313nm的荧光灯管都能够对金黄色葡萄球菌进行灭活,但是在相同光照时间和光照强度下,395±5nm的LED灯珠比波长为309-313nm的荧光灯管的灭活效果更好。
对比三组实验中血小板样品的性质及成分含量,结果如下:
表1血小板保存质量
从表1中的数据可以看到,在利用核黄素光化学法对血小板进行病原体灭活后,各项性质和成分含量的参数与未光照的对照组均有一定的偏离,但是,采用395±5nm的LED灯珠光照对各项性质和成分含量的影响明显小于采用309-313nm的荧光灯管光照对各项性质和成分含量的影响,可见,395±5nm的光照对血小板样品的损伤显著更小。
实施例3
本实施例对相同灭活效果下,采用309-313nm窄谱紫外光的光剂量和395±5nm窄谱紫外光的光剂量进行对比。
具体实验步骤为:
1、取一袋健康献血者血浆(获得当地伦理委员会支持)150ml,加入10uL金黄色葡萄球菌培养液,获得大约4-5log的细菌血浆悬浮液;
2、取9mL细菌血浆悬浮液与1mL生理盐水混合作为对照,放置于4℃冰箱中;
3、然后将500μmol/L的核黄素生理盐水溶液(CAS:83-88-5;购自美国密苏里州圣路易斯Sigma-Aldrich公司)加入细菌血浆悬浮液中,最终核黄素浓度为50μmol/L。
4、分别取300uL加核黄素的细菌血浆悬液转移到无菌24孔板中(孔直径为1.5cm),然后分别暴露于9W 309-313nm荧光灯管(UVB窄带PL-L/PL-S,飞利浦,阿姆斯特丹,荷兰)和1W 395±5nm LED灯珠下,在温度控制的环境中(20-24℃)照射样本。
309-313nm荧光灯管光剂量为9.76J/mL,光照时间为30分钟。395±5nm LED灯珠光剂量为1.25J/mL,光照时间为10分钟。两组实验样本各平行进行6组。
5、光照结束后实验样本和对照样本连续稀释101-106;
6、每个稀释后的样品取100μL加入无菌板中心,每个稀释度样本重复接种8次,测定细菌生长量。
7、37℃生化培养箱(中国上海景洪SHP-080)培养24-48h后,观察各孔细菌生长情况并记录,采用Reed-Muench法计算细菌滴度。
实验结果表明:采用309-313nm紫外光较高光剂量进行照射后与采用395±5nm紫外光较低光剂量进行照射后,309-313nm和395±5nm细菌生长量分别为2.01±1.99log和2.22±1.80log,无统计学差异(P=0.568),即对病原体的灭活效果相当。
从上述实施例中可以看到,本发明优选的波长范围的窄谱紫外光对病原体的灭活效果更好,且对生物液体样本(例如血液产品)中的其他成分的损伤更小。此外,采用发明优选的波长范围的窄谱紫外光,能够选择更短的光照时间和更低的光照能量,从而进一步降低光照对生物液体样本(例如血液产品)中的其他成分的损伤。
Claims (3)
1.一种生物液体样本中病原体的核黄素光化学灭活方法,包括向生物液体样本中加入核黄素并用光辐射生物液体样本的步骤,其特征在于:所述光为波长范围选自390-400 nm范围内的窄谱紫外光;
所述光辐射生物液体样本的光照时间为10-30 min,光照能量范围为0.2-5 J/ml,所述核黄素在生物液体样本中的加入量为40-60 μM;光照环境温度为20-24ºC;
所述生物液体样本为血液产品。
2.按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述光为波峰为395 nm的紫外光。
3.按照权利要求1所述的一种生物液体样本中病原体的核黄素光化学灭活方法,其特征在于:所述血液产品为全血、白细胞减少的全血、包装红细胞、手工血小板、单采血小板、血浆或冷沉淀。
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