基于双分子信标对猪圆环病毒Ⅱ型的简单快速检测

童春义等
摘要根据猪圆环病毒(PCV2)基因组为单链DNA的特点,设计了两条可与PCV2基因组序列特异性杂交的分子信标,建立了基于双分子信标法检测PCV2的方法。实验结果表明,双分子信标法比单分子信标法的灵敏度更高。在10 mmol/L MgCl2、20 mmol/L TrisHCl(pH=80)的杂交缓冲溶液,40 ℃孵育30 min的优化检测体系下,此方法可实现对检测样本在2~200 nmol/L线性范围内的检测,检出限可达1 nmol/L。将双分子信标检测体系用于18例可疑猪瘟样品病毒检测,其中8例呈PCV2阳性,检测结果与PCR法一致,证明此双分子信标法可用于实际猪感染PCV2的诊断。
关键词分子信标; 猪圆环病毒Ⅱ型; 多聚酶链式反应; 定性检测
1引言
分子信标(MB)是一种在目标物不存在时可以形成茎环结构的寡核苷酸探针,探针的5′端和3′端分别标记荧光基团和荧光熄灭基团,正常情况下,荧光基团与荧光熄灭基团靠近发生荧光共振能量转移,导致探针荧光信号淬灭。当溶液中含有能与分子信标环部互补的靶序列时,环部序列与靶分子的结合导致探针结构破坏并恢复荧光\[1\],由于这种检测法无需经过分离多余探针的步骤,且不需要经过传统的PCR扩增过程,不仅被广泛用于复杂均相溶液中的单链DNA、mRNA和microRNA分子的快速、定量检测\[2~6\],也为检测链复杂体系中的单DNA病毒提供了一种可供选择的新工具。
猪圆环病毒Ⅱ型(Porcine circovirus 2,PCV2)是一种呈球形或六角形,无包膜,大小为18~25 nm的ssDNA病毒,被认为是引起断奶仔猪多系统衰竭综合症的主要病原,该病毒感染畜禽后使畜禽的免疫组织细胞受损,导致机体免疫抑制,易并发或继发其它病原感染,最终导致动物死亡并造成重大经济损失\[7\]。因此,开展PCV2的快速、定量检测对于畜禽感染诊断及治疗具有重要的理论意义和实际应用价值。目前,传统的圆环病毒检测主要采用免疫组织化学法、核酸原位杂交检测法\[8,9\]、血清学ELISA检测法\[10\]、PCR检测法、乳胶凝聚法\[11\]等,但这些方法均在一定程度上具有操作复杂、费时费力等缺陷,而且在操作过程中使用的有毒化学物质容易造成环境污染或出现假阳性检测结果。为了在一定程度上克服这些传统方法所具有的缺陷,本研究利用PCV2基因组单链DNA可直接作为分子信标检测对象的特点,设计了两条可单独识别PCV2不同靶点的分子信标,在开展了PCV2 分子信标性能考察和杂交条件优化的基础上,进一步将其用于猪瘟病毒基因组中的PCV2的检测。3结果与讨论
31双分子信标法检测PCV2原理
基于双分子信标(DMB)检测PCV2的原理如图1所示:该检测体系由两条可识别PCV2基因组不同靶点的分子信标(MB1和MB2)、PCV2基因组DNA和杂交缓冲液组成。当溶液中没有PCV2 DNA存在时,维持发夹结构的分子信标不产生荧光信号,加入PCV2 基因组DNA 后,两种分子信标分别与PCV2特异性杂交形成双链结构,破坏分子信标发夹结构的同时伴随荧光信号升高,因此根据荧光信号变化就可以实现溶液中PCV2的快速检测。由于与一种分子信标的结合导致靶分子原有空间结构受到破坏,转变成为易与另一种分子信标发生杂交的结构,从而产生协同效应。如图2所示,本实验将一段PCV2基因组特征片段(cDNA)与分子信标单独和同时作用后,测定荧光信号变化情况。结果表明,单信标作用荧光信号分别增加25和30倍,而双信标作用荧光信号放大近80倍,达到/接近超猝灭分子信标的信号放大能力,说明双信标检测法有助于提高靶分子检测的灵敏度。
为了进一步验证双分子信标法检测结果的可靠性,采用经典的PCR技术对18例样品中的PCV2进行检测,从PCR扩增产物电泳结果(图6)发现:有8例样品出现明显条带,且大小与预期的PCV2产物大小相符(750 bp),说明为阳性样品,有10例没有特异条带,为阴性样品。比对PCR法和双分子信标法检出结果,双分子信标法能够检出浓度、判断为阳性样品的8例均出现明显条带,而判断为阴性样品的10例样品亦未能检出条带,说明双分子信标法测定结果可靠。同时,双分子信标法测定8例阳性样品得出不同样品间PCV2 DNA含量不同,最高的达到65 nmol/L,最小的为25 nmol/L,而对应PCR电泳检测条带亮度不同,浓度大的条带更亮,感染病毒的程度更严重。
4结论
根据猪圆环病毒基因组是单链的特点,设计了两条可特异性识别PCV2基因序列的分子信标,构建了双分子信标检测体系。结果表明,构建的双分子信标检测体系可在2~200 nmol/L范围内对PCV2基因组进行线性检测,最低检测限可达1 nmol/L,可用于临床复杂样品中的PCV2准确检测,表明这种操作简单、快速且成本低的新方法在PCV2的体外检测中具有一定的应用前景。
References
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2Liu B, Wang K M, Xiao Z Q, Wang W, Tan W H, Sun Y, Tang H X, Yang X H Chinese Science Bulletin, 2006, 51(17): 2059-2064
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4Baker M B, Bao G, Searles C D Nucleic Acids Res, 2012, 40(2): e13
5LI Jun, WANG KeMin, TAN WeiHong, LIU Bin, GUO QiuPing,TANG ZhiWen, LIU LingFeng Chem J Chinese Universities, 2004, 25(3): 421-424
李 军, 王柯敏, 谭蔚泓, 刘 斌, 郭秋平, 唐志文, 刘凌风 高等学校化学学报, 2004, 25(3): 421-424
6Yin B C, Liu Y Q, Ye B C J Am Chem Soc, 2012, 134(11): 5064-5067
7LIU ZhengFei Progress in Veterinary Medicine, 2002, 33(2): 14-16
刘正飞 动物医学进展, 2002, 33(2): 14-16
8McNeilly F, Kennedy S, Moffett D, Meehan B M, Foster J C, Clarke E G, Ellis J A, Haines D M, Adair B M, Allan G M J Virol Methods, 1999, 80(2): 123-128
9Harms P A, Sorden S D, Halbur P G, Bolin S R, Lager K M, Morozov I, Paul P S Vet Pathol, 2001, 38(5): 528-539
10Brunborg I M, Fossum C, Lium B, Blomqvist G, Merlot E, Jrgensen A, EliassonSelling L, Rimstad E, Jonassen CM, Wallgren P Acta Vet Scand, 2010, 52(1): 22
11ZHANG HuiMin, YANG Li, LI GuangLiang, WANG Lin, SEHNG ZongHai, HAN HeYou Chinese J Anal Chem, 2011, 39(7): 1113-1116
张慧敏, 杨 利, 李广良, 王 琳, 盛宗海, 韩鹤友 分析化学, 2011, 39(7): 1113-1116
AbstractA doublemolecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection Two singlestranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon Under the optimal conditions of 10 mmol/L MgCl2, 20 mmol/L TrisHCl (pH=80), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method
KeywordsMolecular beacons; Porcine circovirus 2; Polymerase chain reaction; Qualitative detection
5LI Jun, WANG KeMin, TAN WeiHong, LIU Bin, GUO QiuPing,TANG ZhiWen, LIU LingFeng Chem J Chinese Universities, 2004, 25(3): 421-424
李 军, 王柯敏, 谭蔚泓, 刘 斌, 郭秋平, 唐志文, 刘凌风 高等学校化学学报, 2004, 25(3): 421-424
6Yin B C, Liu Y Q, Ye B C J Am Chem Soc, 2012, 134(11): 5064-5067
7LIU ZhengFei Progress in Veterinary Medicine, 2002, 33(2): 14-16
刘正飞 动物医学进展, 2002, 33(2): 14-16
8McNeilly F, Kennedy S, Moffett D, Meehan B M, Foster J C, Clarke E G, Ellis J A, Haines D M, Adair B M, Allan G M J Virol Methods, 1999, 80(2): 123-128
9Harms P A, Sorden S D, Halbur P G, Bolin S R, Lager K M, Morozov I, Paul P S Vet Pathol, 2001, 38(5): 528-539
10Brunborg I M, Fossum C, Lium B, Blomqvist G, Merlot E, Jrgensen A, EliassonSelling L, Rimstad E, Jonassen CM, Wallgren P Acta Vet Scand, 2010, 52(1): 22
11ZHANG HuiMin, YANG Li, LI GuangLiang, WANG Lin, SEHNG ZongHai, HAN HeYou Chinese J Anal Chem, 2011, 39(7): 1113-1116
张慧敏, 杨 利, 李广良, 王 琳, 盛宗海, 韩鹤友 分析化学, 2011, 39(7): 1113-1116
AbstractA doublemolecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection Two singlestranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon Under the optimal conditions of 10 mmol/L MgCl2, 20 mmol/L TrisHCl (pH=80), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method
KeywordsMolecular beacons; Porcine circovirus 2; Polymerase chain reaction; Qualitative detection
5LI Jun, WANG KeMin, TAN WeiHong, LIU Bin, GUO QiuPing,TANG ZhiWen, LIU LingFeng Chem J Chinese Universities, 2004, 25(3): 421-424
李 军, 王柯敏, 谭蔚泓, 刘 斌, 郭秋平, 唐志文, 刘凌风 高等学校化学学报, 2004, 25(3): 421-424
6Yin B C, Liu Y Q, Ye B C J Am Chem Soc, 2012, 134(11): 5064-5067
7LIU ZhengFei Progress in Veterinary Medicine, 2002, 33(2): 14-16
刘正飞 动物医学进展, 2002, 33(2): 14-16
8McNeilly F, Kennedy S, Moffett D, Meehan B M, Foster J C, Clarke E G, Ellis J A, Haines D M, Adair B M, Allan G M J Virol Methods, 1999, 80(2): 123-128
9Harms P A, Sorden S D, Halbur P G, Bolin S R, Lager K M, Morozov I, Paul P S Vet Pathol, 2001, 38(5): 528-539
10Brunborg I M, Fossum C, Lium B, Blomqvist G, Merlot E, Jrgensen A, EliassonSelling L, Rimstad E, Jonassen CM, Wallgren P Acta Vet Scand, 2010, 52(1): 22
11ZHANG HuiMin, YANG Li, LI GuangLiang, WANG Lin, SEHNG ZongHai, HAN HeYou Chinese J Anal Chem, 2011, 39(7): 1113-1116
张慧敏, 杨 利, 李广良, 王 琳, 盛宗海, 韩鹤友 分析化学, 2011, 39(7): 1113-1116
AbstractA doublemolecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection Two singlestranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon Under the optimal conditions of 10 mmol/L MgCl2, 20 mmol/L TrisHCl (pH=80), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method
KeywordsMolecular beacons; Porcine circovirus 2; Polymerase chain reaction; Qualitative detection
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