AMP法检测儿童粪便中肠道腺病毒的建立
赵娜1, 刘金霞1, 孙殿兴2
1.承德医学院病原生物学实验中心,承德 067000
2.中国人民解放军白求恩国际和平医院全军肝病诊治中心,石家庄 050082
孙殿兴,Email:sundianxing@hotmail.com
摘要

目的通过应用环介导等温扩增(LAMP)建立一种快速,简便的可视化检测儿童肠道腺病毒核酸的方法.方法登录GenBank网站下载腺病毒40和41基因型的六邻体序列,根据该区段的保守序列设计4条LAMP引物.将反应体系加入EP管,在恒温条件(65 ℃)扩增60 min后,再以凝胶电泳和钙黄绿素染色评价LAMP法的特异性和灵敏度,并和PCR法的结果相比较.最后,同时用LAMP和PCR法对40份可疑临床样本进行检测,应用卡方检验进行统计学分析.结果LAMP法能准确地使腺病毒40和41型得到扩增,酶切结果也正确,显示了LAMP法较好的特异性.LAMP的灵敏度比PCR高约10倍.经过对40份临床样本的检测,LAMP与PCR均没有发生非特异扩增,LAMP法和PCR法的一致性为92.5%( P>0.05)且LAMP法没有漏检阳性样本.另外,使用钙黄绿素染色的方法更好地显示了产物检测的可视性和简便性.结论该研究初步建立了快速,简便,可视化检测肠道腺病毒的LAMP技术,在基层医疗机构有一定的实用价值.

关键词: 肠道腺病毒; 环介导等温扩增(LAMP); 儿童
中图分类号:R373 文献标志码:A 文章编号:1002-2694(2016)02-0124-04
Detection for enteric adenovirus in children by a loop-mediated isothermal amplification method
ZHAO Na1, LIU Jin-xia1, SUN Dian-xing2
1.Chengde Medical University, Chengde 067000, China
2. Bethune International Peace Hospital, Shijiazhuang 050082, China
Sun Dian-xing, Email: sundianxing@hotmail.com; Liu Jin-xia, Email: 1158210524@qq.com
Abstract

We aimed to establish a rapid, simple and visualized nucleic acids detection method for enteric adenovirus in children by loop-mediated isothermal amplification(LAMP). Firstly, we logged in GenBank and downloaded the hexon genetic region of adenovirus genotypes 40 and 41. Four LAMP primers were designed according to its conserved region. The amplification was completed by incubating all reagents in an EP tube under the isothermal condition (65 ℃) for 60 min. Then, the specificity and sensitivity of LAMP method were evaluated by both gel electrophoresis and calcein-stain methods, and the results of the method were compared with that of PCR. At last, A total of 40 specimens collected from suspicious children were simultaneously detected by both LAMP and PCR methods, statistically significant analysis was evaluated by using chi-square test. Results showed that LAMP method could accurately identify adenovirus genotypes 40 and 41, and the results of digestive production by restriction enzyme were right as well, which showed good specificity. The sensitivity of LAMP method was about tenfold higher than that of PCR. After detection of 40 clinical samples, non-specific products could not be detected by LAMP and PCR methods. Compared with PCR, LAMP exhibited 92.5% ( P>0.05) identity during the detection of enteric adenovirus without the missing of positive samples. In addition, the calcein-stain method for products detection showed good visibility and simplicity. In conclusion, our study established a rapid, simple and visualized detection method for enteric adenovirus by LAMP, which has some practical value in primary care hospitals.

Keyword: enteric adenovirus; loop-mediated isothermal amplification(LAMP); children

人类腺病毒属于双链DNA病毒, 具有对称的20面体衣壳, 无包膜, 包含240个六邻体和12个五邻体, 共57个血清型[1].腺病毒分为A~G共7个亚属组, 其中F组40和41型, 又称肠道腺病毒, 是仅次于轮状病毒的引起5岁以下儿童腹泻的主要病原体之一[2, 3, 4].

然而, 目前检测肠道腺病毒的方法存在不足之处:电镜法灵敏度较低, 单克隆抗体为基础的ELISA[5].培养困难[6], PCR法易造成交叉污染而导致假阳性[2].实时荧光定量PCR技术具有良好的灵敏度和特异度, 且不需开盖检测扩增产物, 从而减少了污染机会, 但是这对操作者的技术和仪器的精密度要求均很高[7].特研究并评价LAMP法的特异性, 灵敏性和临床实用性, 初步建立可视化检测儿童肠道腺病毒40和41型的LAMP技术.

1 材料与方法
1.1 模板来源

标准病毒株(腺病毒F组40和41型, B组3型和7型, D组37和50型)由中国检验检疫科学研究院提供.选取2015年3月间到白求恩国际和平医院就诊的5岁以下儿童, 每天排便3次或3次以上且粪便性状为水样便, 蛋花样便或粘液便作为纳入标准.此期间共采集了40份粪便, 粪便收集后24 h内送实验室, 以适量pH=7.4的PBS悬浮后, 5 000 r/min离心约5 min, 取上清液.按照QIAmp DNA Stool Mini Kit(QIAGEN公司)说明书提取DNA模板, 提取好的DNA进行分装, 冻存于-80 ℃ 冰箱备用.

1.2 LAMP引物的设计

登录NCBI网站, 下载腺病毒40型和41型六邻体(Hexon)区段序列, 使用ClusawX软件进行多重序列对比, 确定引物设计区段.根据腺病毒的40型(参考序列Genbank号:AB330121)和41型(参考序列Genbank号:AB330122)的Hexon保守区段, 以LAMP引物软件Primer Explorer V4(http://primerexplorer.jp/e/)设计通用引物.引物的相对位置见图1, 序列见表1, 其中F3和B3为外引物, FIP和BIP为内引物, 所有引物由上海生工公司合成.

图1 LAMP引物的相对位置Fig.1 Relative locations of LAMP primers

表1 LAMP的引物序列 Tab.1 Sequences of LAMP primers
1.3 LAMP反应体系

内引物(FIP和BIP)各1.6 μ mol/L, 外引物(F3和B3)各0.2 μ mol/L, 甜菜碱0.8 mol/L, MgSO4 6 mmol/L, dNTPs 1.4 mmol/L, DNA模板2 μ L, Bst DNA聚合酶8U, 10× 反应缓冲液 2.5 μ L.扩增温度为65 ℃ , 扩增时间为60 min.扩增完成后, 如果体系中提前加入1 μ L的钙黄绿素混合溶液(钙黄绿素和MnCl2的终浓度分别5 mmol/L和10 mmol/L), 肉眼观察到绿色为阳性, 橙色为阴性.取5 μ L LAMP产物上样于2%琼脂糖凝胶中电泳约40 min, 凝胶成像系统下检视阶梯状扩增条带以验证钙黄绿素的染色结果.

1.4 PCR反应体系

各1.0 μ mol/L的引物(引物参考眀红霞等的报道[11]), Taq DNA聚合酶1.25 U, 10× Taq Buffer 5 μ L, 各0.2 mmol/L的dNTPs, 4 mmol/L的MgCl2, 2 μ L模板DNA, 用双蒸水补足50 μ L.将反应体系置于PCR扩增仪上, 设置参数为:95 ℃ 预变性3 min, 95 ℃ 变性30 s, 55 ℃ 退火30 s, 72 ℃ 延伸30 s, 将变性, 退火和延伸步骤循环35次, 最后72 ℃ 再延伸5 min.取5 μ L产物上样于2%琼脂糖凝胶中电泳, 凝胶成像系统下观察扩增条带, 产物大小应为163 bp.

1.5 LAMP的特异性

分别以腺病毒F组(40和41型), B组(3和7型)和D组(37和50型)标准毒株进行LAMP扩增, 将F组扩增产物用内切酶BalI酶切, 37 ℃ 孵育过夜, 预计酶切后片段主要为一条191 bp的条带.实验重复3次以上.

1.6 LAMP的灵敏度

对标准毒株的DNA模板用双蒸水对其进行一系列10倍稀释(稀释度从1∶ 1到1∶ 10 000), 分别进行LAMP和PCR扩增, 比较检测灵敏度.实验重复3次以上.

1.7 统计分析

对40份疑似肠道腺病毒感染的粪便DNA提取物样本分别用LAMP和PCR法检测, 应用SPSS 17.0软件进行卡方检测, P< 0.05具有统计学意义.

2 结果
2.1 LAMP的特异性

F组(40和41型酶切结果如图2A第1, 2泳带所示, 腺病毒40和41型DNA模板酶切后的电泳条带和预计条带相符(191 bp).所有病毒DNA的LAMP扩增结果如图2A第3~8泳带所示, 只有腺病毒40和41型被扩增, B组和D组腺病毒没有被扩增, 该实验进一步说明引物的特异性良好.另外, 由图2B可知, 钙黄绿素的可视化效果和电泳结果相当.

图2 LAMP检测肠道腺病毒的特异性Fig.2 Specificity of LAMP for detection of enteric adenovirus

2.2 LAMP和PCR的灵敏度

将肠道腺病毒标准株用双蒸水进行一系列10倍稀释, 对稀释的模板DNA分别用LAMP和PCR扩增.PCR扩增结果如图3A所示, 随着稀释度的逐渐增大, 扩增条带逐渐减弱, 最终只能检测到的模板稀释度为1∶ 100.用LAMP法的电泳扩增结果如图3B所示, 随着稀释度的逐渐增大, 最终可检测到的模板稀释度为1∶ 1 000.钙黄绿素染色效果如图3C所示, 可用肉眼观察LAMP扩增结果, 其可视化效果和电泳检测的效果相当.

图3 PCR和LAMP检测肠道腺病毒的灵敏度Fig.3 Sensitivity of PCR and LAMP for detection of enteric adenovirus

2.3 LAMP和PCR对临床样本的检测

为确证LAMP诊断临床样本的准确性, 对40份可疑样本分别进行LAMP和PCR扩增并测序.结果LAMP法检测出22份阳性, PCR法检测出19份阳性, 二者的一致率为92.5%, 卡方检验结果P=0.25(P> 0.05), 证明无统计学意义.在PCR法得出阳性结果的样本中, 用LAMP法没有出现得出阴性结果的情况.对结果不一致的3份样本测序, 结果均为腺病毒41型, 这进一步显示了LAMP具有更高的灵敏度.还可确定LAMP和PCR法的阳性率分别为55%和47.5%.

3 讨论

在致病的腺病毒中, 特定的血清型同疾病的症状, 流行分布及高危人群具有一定相关性[12, 13].比如儿童呼吸道感染与腺病毒3, 7和14型相关[14], 角膜结膜炎与腺病毒53和54型相关[15], 肠道腺病毒与腺病毒40和41型相关, Ziros等[16]也用LAMP法进行研究, 与本研究不同的是Ziros等是用两套LAMP引物对肠道腺病毒进行分型检测, 而且产物检测没有达到可视性.

LAMP的特异性验证用不同的腺病毒标准毒株为模板进行扩增, 扩增结果和扩增产物的酶切结果均证明了引物的特异性较好.从原理上解释, 这4条LAMP引物需要同时识别出靶序列的6段特定区段才启动扩增, 而PCR是用两条引物同时识别靶序列的2段特定区段.在灵敏度方面, LAMP的灵敏度比PCR更高; 另外, 常规提取方法, 尤其是从粪便中得到的DNA中可能伴有DNA聚合酶抑制物等杂质, 这对PCR有抑制作用, 而LAMP法的耐干扰性强, 这也是LAMP法比PCR法灵敏的原因之一.

在产物检测阶段用染料钙黄绿素进行可视化检测, 基本原理为反应前与Mn2+结合处于淬灭状态, 反应中副产物焦磷酸根与Mn2+结合释放钙黄绿素, 钙黄绿素的淬灭状态解除, 发出绿色荧光[17], 这种荧光用肉眼可进行结果判断, 简单方便.

LAMP法由于具有优于其他检测方法的优势而正在被广泛的研究应用, 但是该方法也存在不足之处, 比如引物设计繁琐, 易产生气溶胶污染等.因此, 要在大规模样本验证后使其发展成一种经济, 简便的肠道腺病毒检测技术, 便于在基层医院检测或做流行病学调查.

The authors have declared that no competing interests exist.

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