ZNA探针在沙眼衣原体核酸定量检测中的研究

Research on ZNA probe in the quantitative detection of chlamydia trachomatis nucleic acid

目的:探讨ZNA (ZIP Nucleic Acid)探针的PCR性能及其在沙眼衣原体(CT)核酸定量检测中的研究。方法:使用CT阳性质粒,比较偶联不同ZIP数量的ZNA探针的PCR扩增曲线差异;比较ZNA探针与29mer普通Taqman探针(DNA长探针)、20mer普通Taqman探针(DNA短探针)、MGB探针在PCR扩增曲线、反复冻融稳定性及低浓度质粒检出率的差异;使用CT阳性临床样本,比较ZNA探针与DNA长探针、DNA短探针、MGB探针扩增曲线差异及低浓度样本检出率的差异。结果:(1)偶联5个ZIP单位的ZNA探针Ct值和荧光值最优,与偶联1个ZIP的探针相比:Ct值提升1.34 (灵敏度提升2.37倍),荧光值提升30%。(2)偶联5个ZIP的ZNA探针的扩增效率是优选的普通Taqman探针和MGB探针的2.14~2.64倍,荧光值高17%~90%。(3)四组探针冻融稳定性结果显示,ZNA探针的稳定性最佳,低浓度(1×10 4拷贝/mL)的Ct值偏离最小(CV%= 1.4),优于DNA长探针、DNA短探针、MGB探针(CV%= 1.7~3.7)。(4)用35例CT临床样本比较四组探针(DNA长探针、DNA短探针、MGB探针、ZNA探针)的PCR扩增性能,相对于其他三组探针,ZNA探针的扩增灵敏度分别提升1.60倍、0.99倍和1.06倍,且Ct值一致性分析结果显示,ZNA探针对临床样本的检出性能提升更优。(5)使用低浓度质粒模板(分别为200、100、50和10拷贝/mL)比较四组探针的扩增灵敏度,ZNA探针的检出率均优于DNA长探针、DNA短探针、MGB探针;在最低浓度10拷贝/mL时,其他三组探针的检出率只有15%~20%,ZNA探针仍有30%。(6)在20例不同低浓度(分别为200、150、100和50拷贝/mL)临床样本中ZNA探针检出率最高,分别为100%、95%、90%和70%。 结论:偶联5个ZIP的ZNA探针的扩增效率、荧光值、冻融稳定性、临床样本的扩增性能及低浓度样本检出灵敏度都优于普通Taqman探针和MGB探针,ZNA探针可作为一种设计灵活和合成易得的新探针技术,在基因表达领域可进一步提升检测的灵敏度及低浓度样本的检出率。

Objective To investigate the performance of ZNA(ZIP Nucleic Acid)probes and its application in the quantitative detection of Chlamydia trachomatis(CT)nucleic acid.Methods Use CT positive plasmids to compare the PCR amplification curves of ZNA probes coupled with different ZIP numbers.Compare ZNA probes with other three sets of probes[namely,29mer ordinary Taqman probes(long-DNA probe),20mer ordinary Taqman probes(short-DNA probe)and MGB probes]for stability in PCR amplification curves and repeated freezing and thawing,and the difference in the detection rate of low-concentration plasmids.Use CT positive clinical samples to compare the difference in amplification curves between ZNA probes,long-DNA probe,short-DNA probe and MGB probes,and the detection rate of low-concentration samples.Results(1)The Ct value and fluorescence value of the probes coupled with 5ZIP units are both better than those coupled with a smaller number of ZIPs.And the difference is biggest when compared with only coupled with 1 ZIP unit:Ct value increased by 1.34(sensitivity increased by 2.37 times),and fluorescence value increased by 30%.(2)The amplification efficiency of the ZNA probe coupled with 5 ZIPs is 2.14-2.64 times that of the preferred ordinary Taqman probe and MGB probe,and the fluorescence value is 17%-90%higher.(3)The probe freeze-thaw stability results show that the ZNA probe has the best stability,and the lowest concentration of Ct value has the smallest deviation(CV%=1.4),which is better than the other three sets of probes(CV%=1.7-3.7).(4)Using 35 CT positive clinical samples to compare the PCR amplification performance,compared with other three sets of probes,the amplification sensitivity of ZNA probes was increased by 1.60,0.99 and 1.06 times respectively.And the results of the consistency analysis of the Ct value show that compared with short-DNA probe and MGB probes,ZNA probes have better detection performance for clinical samples.(5)Use low concentration plasmid template(200,100,50 and 10 copies/mL respectively)to compare the amplification sensitivity of the four sets of probes,the detection rate of ZNA probe is the best.Especially,at the lowest concentration 10 copies/mL,the detection rate of the other sets of probes is only 15%-20%,but the ZNA probe is still 30%.(6)In 20 clinical samples with different low concentrations(200,150,100,and 50 copies/mL),the detection rate of ZNA probes was the highest,which were 100%,95%,90%,and 70%,respectively.Conclusions Through testing of the amplification efficiency,fluorescence value,freeze-thaw stability,the amplification performance of clinical samples and the detection sensitivity of low-concentration samples,ZNA probes coupled with 5 ZIPs show better performance than ordinary Taqman probes and MGB probes.As a new probe technology with flexible design and easy synthesis,ZNA probe can further improve detection sensitivity of low concentration samples in the field of gene expression.