丝虫SYBR Green实时荧光PCR检测方法的建立

Establishment of a SYBR Green real-time fluorescent PCR assay for the detection of filarial

目的 建立一种快速、灵敏的丝虫SYBR Green实时荧光聚合酶链式反应(PCR)检测方法,为丝虫病的快速筛查和临床诊断提供实验室依据。方法 根据丝虫基因组保守区域,设计并筛选特异性引物。对引物浓度、荧光染料浓度及扩增条件进行优化,建立丝虫SYBR Green实时荧光PCR检测方法,并对其检测下限、特异性、抗干扰能力、重复性和亚型检测能力等进行评估。结果 本研究建立的丝虫SYBR Green实时荧光PCR检测方法能对感染人的5种丝虫(班氏丝虫、马来丝虫、盘尾丝虫、罗阿丝虫、帝汶丝虫)进行特异性扩增,与其他相近寄生虫(锥虫等)无交叉反应。该方法的检测下限为500 fg,批内精密度测试循环阈值(Ct值)为32.37~33.03,均值为32.73,标准差为0.20,变异系数(CV)为0.61%。干扰试验中,存在干扰因素样本的Ct值与正常样本的Ct值差值分别为0.79(溶血)、0.78(脂血)、0.29(乳糜尿)、0.51(腐败蚊体),表明该方法能有效对抗溶血、脂血、乳糜尿、腐败蚊虫组织等干扰因素的影响。结论 本研究建立的SYBR Green实时荧光PCR方法具有特异性强、敏感度高、重复性好以及抗干扰能力强的优点,是一种快速检测丝虫的可行方法。

Objectives To establish a SYBR Green real-time fluorescent polymerase chain reaction(PCR) which can detect filarial rapidly and sensitively. To provide laboratory basis for rapid screening and clinical diagnosis of filariasis. Methods Specific primers were designed to target the conserved region of filarial genome and then the real-time fluorescent PCR was established by optimizing both the concentration of SYBR Green, primers and the amplification conditions. The lower limit of detection, specificity, repeatability, anti-interference ability and subtype detection ability of the newly established assay were evaluated. Results The established SYBR Green real-time fluorescent PCR assay for the detection of five kinds of filarial, including Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus, Loa loa, and Brugia tinori, was specific, without cross-reaction to other related pathogens, including trypanosome. The lower limit of detection of the assay could reach as low as 500 fg per reaction, indicating its high sensitivity. The assay also showed high repeatability. During within batch testing, the cycle threshold(Ct) values were between 32.37 and 33.03, average Ct value was 32.73, SD was 0.20, and the coefficient of variation(CV) was 0.61%. In the interference experiment, the Ct value difference between samples with interference factors and normal samples was 0.79(hemolysis), 0.78(lipid blood), 0.29(chyluria), and 0.51(decayed mosquito body), respectively, indicating that it can effectively combat the influence of interference factors such as hemeferroheme, lipids, chyluria, and decayed mosquito tissue. Conclusion The established SYBR Green real-time fluorescent PCR assay showed high sensitivity, specificity, repeatability and anti-influence ability, which could provide a rapid method in the detection of filarial.