背景与目的

针对核酸进行检测的分子生物学方法在个体化精准医疗中的重要作用日益凸显，无论是在遗传水平上相关基因的检测，或是在新发传染病中的大规模筛查，都是对大众健康的有力保障。基于TaqMan探针的PCR扩增技术是当前分子诊断技术中的重要基础，但核酸提取是阻碍通量提高、制约现场运用的关键问题，其过程中DNA或RNA的随机断裂丢失也影响其检测的灵敏度和定量准确性，针对RNA的检测还需进行反转录导致检测成本和步骤增加。此外，针对不同核酸序列设计的TaqMan探针只能用于特定靶标的检测，更换靶标时需要重新设计，对于经常需要PCR检测的实验室而言相当浪费。基于此，本研究拟采取直接扩增或靶标富集的免提核酸技术，结合不同信号的TaqMan探针，开发一种仅需采集口腔拭子即可进行检测的脂质代谢单核苷酸多态性分型技术，以及一种基于捕获和连接的两重TaqMan-PCR（capture and ligation TaqMan probe-PCR, CLIP-TaqMan）技术用以鉴别诊断疟疾/巴贝斯虫病和测量端粒的相对长度，以实现高通量、低成本和快速简便检测各类DNA或RNA样品的目的。

方法

本研究将直接PCR与TaqMan探针相结合，使用无创性的口腔拭子作为检材，将口腔拭子放入样品处理液后进行短暂震荡和离心，取少量上清进行直接qPCR扩增分析，采用提取的DNA对设计的TaqMan探针进行PCR退火延伸温度和扩增程序的优化，探索了不同样品处理液、探针冻融稳定性和样品保存时间对分型结果的影响，完善方法后对42份口腔拭子样品进行了验证实验。将基于捕获和连接的PCR技术与TaqMan探针相结合，对巴贝斯虫体外转录RNA和实际血液样品进行检测，评估了方法的特异性和重复性；并通过多个不同荧光基团修饰的TaqMan探针，达到两重检测的目的，对巴贝斯虫和疟原虫的模拟混合样品进行鉴别诊断。同时，将两重CLIP-TaqMan技术运用于检测端粒和单拷贝基因，建立了DNA浓度和Ct值的标准曲线，以此建立一种端粒长度的相对定量方法，以实际血液样品初步确定了端粒长度与年龄的对应关系，并与已有方法进行了相关性分析。

结果

本方法仅需对口腔拭子样本简单处理后即可进行qPCR分析，并且可在室温情况下保存4 d。优化后的方法能够较好地区分rs688和rs964184位点的野生纯合子、突变纯合子及杂合子。CLIP-TaqMan技术检测巴贝斯虫时，优化后的方法检测恶性疟原虫、间日疟原虫、寨卡病毒和登革热病毒时均无信号，批次内和批次间重复性小于2%，检测10⁵和10³个感染红细胞接种量的小鼠模型在接种4 h后即有阳性结果，而显微镜检查分别在第3 d或第8 d方可观察到被感染的红细胞。两重检测时在体外转录模拟样品和混合血液模拟样品中均可对2种寄生虫进行检测，并且与单重检测的Ct值一致。CLIP-TaqMan检测端粒长度时，优化后的探针用量为0.04 nmol/L，4例实际样品的检测大致表现出了随年龄增长端粒变短的趋势，与已有方法的相关性分析R²=0.9436。

结论

本研究建立了基于直接TaqMan-PCR的SNP分型新技术，该技术快速便捷、通量高且成本低，为大规模筛查携带易感基因的风险人群提供了高效且准确的新方法；同样建立了基于捕获和连接的通用TaqMan-PCR技术，实现了巴贝斯虫病和疟疾的鉴别诊断，并可用于端粒相对长度的测量，在分子诊断领域有良好的应用前景。

Objective

Molecular biology methods targeting nucleic acids play an increasingly important role in individualized precision medicine, either in the detection of relevant genes at the genetic level or in the mass screening of emerging infectious diseases, which can be seen as a powerful safeguard for public health. TaqMan probe based PCR amplification is an important foundation of current molecular diagnostic technology, but the nucleic acid extraction process is a key problem that hinders the improvement of throughput and limits the field application. The loss of random breaks of DNA or RNA during the process also affects the sensitivity and accuracy of quantification. At the same time, the detection of RNA also needs to be performed by reverse transcription, which leads to an increase in the cost of the test and the number of steps. In addition, the design of TaqMan probes for different nucleic acid sequences can only be used for the detection of specific targets, and it needs to be redesigned when changing targets, which is quite wasteful for laboratories that often need PCR detection. In this study, some techniques have been, such as proposed to adopt hands-free nucleic acid technique of direct amplification or target enrichment and combine with TaqMan probes with different signals, to develop a single-nucleotide polymorphism typing technique for lipid metabolism that can be detected only by collecting oral swabs, as well as develop a double-plex capture and ligation TaqMan-PCR (CLIP-TaqMan) for the differential diagnosis of Malaria/Babesiosis and the measurement of the relative length of telomeres for high throughput, low cost, rapid and convenient assay to detect various DNA or RNA samples.

Methods

In this study, direct PCR was combined with TaqMan probes, and non-invasive oral swabs were used as test materials. The oral swabs were placed into the sample treatment solution and then briefly shaken and centrifuged, and a small amount of the supernatant was taken for the direct qPCR amplification analysis. Extracted DNA was used to optimize the PCR annealing extension temperature and amplification procedure for the designed TaqMan probe. The effects of different sample treatment solutions, the freeze-thawing stability of probe and the effects of sample storage time on genotyping results were explored, and validation experiments were carried out on 42 oral swab samples after refining the method. We combined capture and ligation probe-PCR with TaqMan probes for detection of Babesia in vitro transcribed RNA and blood samples, and the specificity and reproducibility of the method were evaluated. By using multiple TaqMan probes modified with different fluorescent moieties, we achieved double-plex detection for differential diagnosis of simulated mixed samples of Babesia and Plasmodium. Meanwhile, the double-plex CLIP-TaqMan assay was applied to detect telomere and single-copy gene, and standard curves of DNA concentration and Ct value were established, so as to establish a relative quantitative method of telomere length. The correspondence between telomere length and age was preliminarily determined with blood samples, and correlation analysis were conducted with the existing method.

Results

The technology requires only simple treatment of oral swab for PCR analysis and can be stored at room temperature for 4 d. The optimized method is able to distinguish homozygous wild type, homozygous mutant type and heterozygotes at rs688 and rs964184 loci. CLIP-TaqMan for detecting Babesia was no signal in the detection of Plasmodium falciparum, P. vivax, Zika virus and dengue virus after optimization, and intro- and inter-variability of this method less than 2%. The detection of 10⁵ and 10³ infected erythrocyte inoculums in the mouse model gave positive results 4 h after inoculation, whereas infected erythrocytes could not be observed by microscopy examination until the 3rd or 8th day, respectively. The two parasites could be detected in both IVT and mixed blood sample in the double-plex assay, and the Ct values were consistent with those of the single-plex assay. The optimized probe dosage of 0.04 nmol/L for telomere length detection by CLIP-TaqMan in the four blood samples broadly showed a trend of shorter telomeres with age, and the correlation analysis with the established methods showed a correlation value of R²=0.9436.

Conclusion

A fast, convenient, high-throughput, and low-cost SNP genotyping method has been established, providing an efficient and accurate new approach for large-scale screening of high-risk populations carrying susceptibility genes. We also established CLIP-TaqMan assay to achieve differential diagnosis of Babesia and Malaria. And it can also be used for the measurement of the relative length of telomeres, which has potential application value in the field of molecular diagnosis.