DNA功能化金纳米颗粒在沙眼衣原体检测中的应用
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摘要
目的建立一种基于DNA功能化金纳米颗粒检测沙眼衣原体的新方法。方法分析沙眼衣原体(Chlamydia trachomatis,Ct)Trp B保守基因序列,设计2条特异性寡核苷酸链,一条进行巯基修饰后与柠檬酸钠还原法制备的金纳米颗粒结合得到信号探针,另一条用生物素标记作为捕获探针。制备的靶序列与两种探针在包被链霉亲和素的酶标板内形成牢固的带有金纳米颗粒的核酸复合物,加入银染液后,经酶标仪读取630 nm处A值或肉眼观察96孔板底灰度。对建立的检测体系进行特异性和灵敏度验证。用建立的检测体系及荧光定量PCR法对可疑临床沙眼衣原体样本进行检测。结果制备的金纳米颗粒经透射电子显微镜(transmission electron microscope,TEM)观察确定其粒径约20 nm,吸收峰为518 nm,金纳米颗粒标记的信号探针吸收峰为520 nm;不同靶序列浓度的银染结果可用肉眼进行分辨。优化后的检测体系为:反应时间2 h,杂交温度25℃,生物素探针浓度100 nmol/L,银染色时间8 min。建立的方法能特异性检测沙眼衣原体核酸序列,与其他生殖道病原菌无交叉反应,最低检测浓度达55 pmol/L。49份临床样本检出率为61%,与荧光定量PCR法相比,差异无统计学意义(P>0.05)。结论建立了一种简便、高效、特异的沙眼衣原体检测方法,成本低廉,为沙眼衣原体的临床病原学诊断提供了新的途径。
Objective To establish a new method for detection of Chlamydia trachomatis with DNA functionalized gold nanoparticles. Methods Two specific oligonucleotide chains were designed according to the Trp B gene sequence of C. trachomatis,of which one was combined with gold nanoparticles obtained by sodium citrate reduction method after thiol modification and used as the signal probe,while the another one was labeled with biotin and used as the capture probe. The prepared target sequence and the two probes formed a strong complex with gold nanoparticles on the microplate coated with streptomycin. After addition of silver staining solution,the result was obtained by reading the A630 value with microplate reader or by visual observation of gray scale with naked eye. The established detection system was verified for specificity and sensitivity,and used for detection of clinical samples of C. trachomatis,serving fluorescent quantitative PCR as control. Results The size of prepared gold nanoparticles was defined as 20 nm by TEM,while the absorption peak appeared at a wavelength of 518 nm. However,the absorption peak of the signal probe labeled by nanoparticles appeared at 520 nm. The silver staining results of various concentrations of target sequence could be distinguished visually. The optimal reaction time,temperature for hybridization,biotin probe concentration and time for silver staining were 2 h,25 ℃,100 nmol/L and 8 min respectively. A highly specific detection method was established,which showed no cross reaction with other reproductive tract pathogens,with a detection limit of 55 pmol/L. The detection rate of 49 clinical specimens was 61%,which showed no significant difference with that by fluorescent quantitative PCR(P > 0. 05). Conclusion A simple,effective,specific and economic method for detection of C. trachomatis was established,which provided a new route for clinical etiological diagnosis of C. trachomatis infection.
Objective To establish a new method for detection of Chlamydia trachomatis with DNA functionalized gold nanoparticles. Methods Two specific oligonucleotide chains were designed according to the Trp B gene sequence of C. trachomatis,of which one was combined with gold nanoparticles obtained by sodium citrate reduction method after thiol modification and used as the signal probe,while the another one was labeled with biotin and used as the capture probe. The prepared target sequence and the two probes formed a strong complex with gold nanoparticles on the microplate coated with streptomycin. After addition of silver staining solution,the result was obtained by reading the A630 value with microplate reader or by visual observation of gray scale with naked eye. The established detection system was verified for specificity and sensitivity,and used for detection of clinical samples of C. trachomatis,serving fluorescent quantitative PCR as control. Results The size of prepared gold nanoparticles was defined as 20 nm by TEM,while the absorption peak appeared at a wavelength of 518 nm. However,the absorption peak of the signal probe labeled by nanoparticles appeared at 520 nm. The silver staining results of various concentrations of target sequence could be distinguished visually. The optimal reaction time,temperature for hybridization,biotin probe concentration and time for silver staining were 2 h,25 ℃,100 nmol/L and 8 min respectively. A highly specific detection method was established,which showed no cross reaction with other reproductive tract pathogens,with a detection limit of 55 pmol/L. The detection rate of 49 clinical specimens was 61%,which showed no significant difference with that by fluorescent quantitative PCR(P > 0. 05). Conclusion A simple,effective,specific and economic method for detection of C. trachomatis was established,which provided a new route for clinical etiological diagnosis of C. trachomatis infection.
引文
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[12]QUINTELA I A,DE LOS R B,LIN C S,et al.Simultaneous direct detection of Shiga-toxin producing Escherichia coli(STEC)strains by optical biosensing with oligonucleotide-func-tionalized gold nanoparticles[J].Nanoscale,2015,7(6):2417-2426.
[13]TIET P,CLARK K C,MCNAMARA J N,et al.Colorimetric detection of Staphylococcus aureus contaminated solutions without purification[J].Bioconjug Chem,2017,28(1):183-193.
[14]LARGUINHO M,SANTOS S,ALMEIDA J,et al.DNA adduct identification using gold-aptamer nanoprobes[J].IET Nanobiotechnol,2015,9(2):95-101.
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[16]SANG F,LIU J,ZHANG X,et al.An aptamer-based colorimetric Pt(II)assay based on the use of gold nanoparticles and a cationic polymer[J].Mikrochim Acta,2018,185(5):267.DOI:10.1007/s00604-018-2794-6.
[17]TAKAHASHI F,YAMAMOTO N,TODORIKI M,et al.Sonochemical preparation of gold nanoparticles for sensitive colorimetric determination of nereistoxin insecticides in environmental samples[J].Talanta,2018,188:651-657.
[18]IAROSSI M,SCHIATTARELLA C,REA I,et al.Colorimetric immunosensor by aggregation of photochemically functionalized gold nanoparticles[J].ACS Omega,2018,3(4):3805-3812.
[2]SOBINOFF A P,DANDO S J,REDGROVE K A,et al.Chlamydia muridarum infection-induced destruction of male germ cells and sertoli cells is partially prevented by Chlamydia major outer membrane protein-specific immune CD4 cells[J].Biol Reprod,2015,92(1):27.DOI:10.1095/biolreprod.114.124180.
[3]HUFNAGEL K,LUEONG S,WILLHAUCK-FLECKENSTEIN M,et al.Immunoprofiling of Chlamydia trachomatis using wholeproteome microarrays generated by on-chip in situ expression[J].Sci Rep,2018,8(1):7503.DOI:10.1038/s41598-018-25918-3.
[4]TRABERT B,WATERBOER T,IDAHL A,et al.Antibodies against Chlamydia trachomatis and ovarian cancer risk in two independent populations[J].J Natl Cancer Inst,2018,111(2):129-136.DOI:10.1093/jnci/djy084[Epub ahead of print].
[5]AHMADI A,RAMAZANZADEH R,SAYEHMIRI K,et al.Association of Chlamydia trachomatis infections with preterm delivery;a systematic review and meta-analysis[J].BMC Pregnancy Childbirth,2018,18(1):240.DOI:10.1186/s12884-018-1868-0.
[6]WANG Y F,WANG L M,YANG H Y.Evaluation of two methodologies for Chlamydia trachomatis detection in cervical samples of two clinics[J].Natl Med J China,2017,97(17):1330-1332.(in Chinese)王英芳,王莉敏,杨红云.不同科室来源沙眼衣原体宫颈拭子样本的检测分析[J].中华医学杂志,2017,97(17):1330-1332.
[7]SKULSKA E,MLYNARCZYK-BONIKOWSKA B,WALTER DW S,et al.The comparison of real-time PCR and direct immunofluorescence in laboratory diagnostics of chlamydiosis in patients of Department of Dermatology and Venereology Medical University of Warsaw[J].Med Dosw Mikrobiol,2015,67(3-4):173-180.
[8]WANG Q W,ZHONG Q.Application of PCR with fluorescent probe to test for Chlamydia trachomatis,Neisseria gonorrhoeae and Ureaplasma urelyticum in secretion[J].Chin J Biologicals,2018,31(3):307-310,314.(in Chinese)王勤婉,钟琦.PCR荧光探针法在检测分泌物沙眼衣原体、淋球菌及解脲支原体中的应用[J].中国生物制品学杂志,2018,31(3):307-310,314.
[9]HORNER P J,WILLS G S,RIGHARTS A,et al.Chlamydia trachomatis Pgp3 antibody persists and correlates with selfreported infection and behavioural risks in a blinded cohort study[J].PLoS One,2016,11(3):e151497.
[10]KHAN E R,HOSSAIN M A,PAUL S K,et al.Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis[J].Mymensingh Med J,2012,21(2):190-194.
[11]DYKMAN L,KHLEBTSOV N.Gold nanoparticles in biomedical applications:recent advances and perspectives[J].Chem Soc Rev,2012,41(6):2256-2282.
[12]QUINTELA I A,DE LOS R B,LIN C S,et al.Simultaneous direct detection of Shiga-toxin producing Escherichia coli(STEC)strains by optical biosensing with oligonucleotide-func-tionalized gold nanoparticles[J].Nanoscale,2015,7(6):2417-2426.
[13]TIET P,CLARK K C,MCNAMARA J N,et al.Colorimetric detection of Staphylococcus aureus contaminated solutions without purification[J].Bioconjug Chem,2017,28(1):183-193.
[14]LARGUINHO M,SANTOS S,ALMEIDA J,et al.DNA adduct identification using gold-aptamer nanoprobes[J].IET Nanobiotechnol,2015,9(2):95-101.
[15]VEIGAS B,JACOB J M,COSTA M N,et al.Gold on paperpaper platform for Au-nanoprobe TB detection[J].Lab Chip,2012,12(22):4802-4808.
[16]SANG F,LIU J,ZHANG X,et al.An aptamer-based colorimetric Pt(II)assay based on the use of gold nanoparticles and a cationic polymer[J].Mikrochim Acta,2018,185(5):267.DOI:10.1007/s00604-018-2794-6.
[17]TAKAHASHI F,YAMAMOTO N,TODORIKI M,et al.Sonochemical preparation of gold nanoparticles for sensitive colorimetric determination of nereistoxin insecticides in environmental samples[J].Talanta,2018,188:651-657.
[18]IAROSSI M,SCHIATTARELLA C,REA I,et al.Colorimetric immunosensor by aggregation of photochemically functionalized gold nanoparticles[J].ACS Omega,2018,3(4):3805-3812.
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