Luminex液相芯片杂交影响因素探讨
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摘要
目的探讨微珠用量和链霉亲和素-藻红蛋白(SAPE)对Luminex液相芯片杂交结果的影响,并进一步优化杂交条件。方法以鼠伤寒沙门菌过夜培养物原液和107cfu/ml、103cfu/ml菌液为待测物,经多重PCR扩增并标记后,与不同体积微珠混合液和不同浓度SAPE杂交,比较不同杂交条件下的中位荧光强度(median fluorescence intensity,MFI);在此基础上,选择不同用量多重PCR产物、不同杂交温度和时间进行测试,比较其MFI。结果 MFI并不随着微珠用量增加而增强,1 000个每种微珠每反应,即可满足杂交需要;SAPE浓度直接影响样品和背景MFI,当SAPE浓度为5.0μg/ml时,杂交结果较为理想,其杂交信号MFI稳定,且背景信号较低;过量的PCR产物并不利于杂交反应,且应选择最佳杂交温度和时间,5.0μl是较为理想的PCR产物用量,42℃杂交25 min是较为理想的杂交条件。结论为优化Luminex液相芯片杂交,选择适当的微珠用量、SAPE浓度、PCR产物用量及杂交温度和时间是非常必要的。
Objective To study the effects of microsphere dosages and Streptavidin-R-phycoerythrin( SAPE) levels on the hybridization results of Luminex suspension array,and to further optimize the hybridization conditions. Methods After growing overnight,the bacterial culture of Salmonella Typhimurium as well as 103cfu/ml and 107cfu/ml of bacterial liquid were used as specimens. After multiplex-PCR amplification and labeling,the PCR products were hybridized with different volume of mixed microspheres and different concentration of SAPE. The median fluorescence intensity( MFI) under different hybridization conditions was compared. Furthermore,different doses of PCR products,different hybridization temperature as well as different time were tested,and the hybridization results were compared. Results MFI did not increase with the microsphere dosages,and1 000 microspheres of each set were enough for each hybridization reaction. SAPE levels influenced the MFI of specimens and background directly,when the concentration of SAPE was 5. 0 μg/ml,good hybridization was obtained. The hybridization signal MFI is stable and the background signal is low; Excessive PCR products were not good for hybridization,and hybridization should be performed under appropriate temperature and time. 5. 0 μl was an ideal amount of PCR product,and hybridization at42 ℃ for 25 min was ideal. Conclusion To optimize the hybridization conditions of Luminex suspension array,it is necessary to determine the right microsphere dosages,SAPE levels,the amount of PCR products,as well as the optimum temperature and time for hybridization reactions.
Objective To study the effects of microsphere dosages and Streptavidin-R-phycoerythrin( SAPE) levels on the hybridization results of Luminex suspension array,and to further optimize the hybridization conditions. Methods After growing overnight,the bacterial culture of Salmonella Typhimurium as well as 103cfu/ml and 107cfu/ml of bacterial liquid were used as specimens. After multiplex-PCR amplification and labeling,the PCR products were hybridized with different volume of mixed microspheres and different concentration of SAPE. The median fluorescence intensity( MFI) under different hybridization conditions was compared. Furthermore,different doses of PCR products,different hybridization temperature as well as different time were tested,and the hybridization results were compared. Results MFI did not increase with the microsphere dosages,and1 000 microspheres of each set were enough for each hybridization reaction. SAPE levels influenced the MFI of specimens and background directly,when the concentration of SAPE was 5. 0 μg/ml,good hybridization was obtained. The hybridization signal MFI is stable and the background signal is low; Excessive PCR products were not good for hybridization,and hybridization should be performed under appropriate temperature and time. 5. 0 μl was an ideal amount of PCR product,and hybridization at42 ℃ for 25 min was ideal. Conclusion To optimize the hybridization conditions of Luminex suspension array,it is necessary to determine the right microsphere dosages,SAPE levels,the amount of PCR products,as well as the optimum temperature and time for hybridization reactions.
引文
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[11]Lin Z,Wang X,Li ZJ,et al.Development of a sensitive,rapid,biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone[J].Talanta,2008,75(4):965-972.
[12]聂丽菊,许恒毅,叶称连,等.生物素-链霉亲和素介导受体靶向卵巢癌SKOV3细胞量子点体外成像的研究[J].中国肿瘤临床,2014,41(19):1254-1258.
[13]刘玲玲,唐桂芳,李文刚,等.探针的修饰与浓度、杂交温度及时间对基因芯片杂交信号的影响[J].中国兽医杂志,2012,48(11):17-19.
[14]Ge D,Wang X,Williams K,et al.Thermostable DNA immobilization and temperature effects on surface hybridization[J].Langmuir,2012,28(22):8446-8455.
[15]Markegard CB,Gallivan CP,Cheng DD,et al.Effects of Concentration and Temperature on DNA Hybridization by Two Closely Related Sequences via Large-Scale Coarse-Grained Simulations[J].J Phys Chem B,2016,120(32):7795-7806.
[2]Carter JM,Lin A,Clotilde L,et al.Rapid,multiplexed characterization of Shiga Toxin-producing Escherichia coli(STEC)isolates using suspension array technology[J].Front Microbiol,2016,7:439.
[3]Cabral-Castro MJ,Peralta RH,Cavalcanti MG,et al.A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus[J].J Virol Methods,2016,236:18-24.
[4]石莹,田绿波,樊学军,等.5种重要虫媒病毒液相蛋白芯片多重检测方法的建立及应用[J].中国卫生检验杂志,2015,25(21):3604-3607.
[5]Lin A,Nguyen L,Lee T,et al.Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbeadbased suspension array[J].J Microbiol Methods,2011,87(1):105-110.
[6]陈峰,陆青云,吴尔翔,等.悬浮芯片技术与ELISA技术在3种常规病原体检测中的优劣[J].中国卫生检验杂志,2013,23(15):3060-3064.
[7]郑之北,郑伟,濮小英,等.沙门菌H抗原的x TAG法鉴定[J].中华微生物学和免疫学杂志,2016,36(12):942-947.
[8]Angeloni S,Cordes R,Dunbar S,et al.x MAP RR Cookbook:A collection of methods and protocols for developing multiplex assays with x MAP Technology[M].3rded.www.luminexcorp.com:Luminex Corporation,2016:94-116.
[9]Leunissen ME,Dreyfus R,Sha R,et al.Quantitative study of the association thermodynamics and kinetics of DNA-coated particles for different functionalization schemes[J].J Am Chem Soc,2010,132(6):1903-1913.
[10]李义良,易进华,夏佳慧,等.PCR引物浓度对寡核苷酸液相杂交的影响[J].中国医药生物技术,2010,5(2):122-126.
[11]Lin Z,Wang X,Li ZJ,et al.Development of a sensitive,rapid,biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone[J].Talanta,2008,75(4):965-972.
[12]聂丽菊,许恒毅,叶称连,等.生物素-链霉亲和素介导受体靶向卵巢癌SKOV3细胞量子点体外成像的研究[J].中国肿瘤临床,2014,41(19):1254-1258.
[13]刘玲玲,唐桂芳,李文刚,等.探针的修饰与浓度、杂交温度及时间对基因芯片杂交信号的影响[J].中国兽医杂志,2012,48(11):17-19.
[14]Ge D,Wang X,Williams K,et al.Thermostable DNA immobilization and temperature effects on surface hybridization[J].Langmuir,2012,28(22):8446-8455.
[15]Markegard CB,Gallivan CP,Cheng DD,et al.Effects of Concentration and Temperature on DNA Hybridization by Two Closely Related Sequences via Large-Scale Coarse-Grained Simulations[J].J Phys Chem B,2016,120(32):7795-7806.