解脲脲原体和微小脲原体相对定量荧光PCR方法的建立及应用

The establishment and clinical use of a Ureaplasma urealyticum and Ureaplasma parvum relative quantitative PCR assay

目的建立一种含内参基因的解脲脲原体(Uu)和微小脲原体(Up)相对定量的荧光PCR分型方法,克服标本采样差异对检测结果的影响,了解患者脲原体感染的生物分群及病原载量。方法设计Uu和Up检测与分型引物探针,使用人β-globin(BG)作为相对定量内参引物探针,建立Uu、Up和BG的三重荧光PCR方法,使用核酸标准品建立标准曲线,计算宿主细胞中Uu或Up的相对含量,并确定体系Uu和Up检测下限。使用20株Uu,30株Up临床分离株以及22种生殖道常见病原体进行体系灵敏度和特异性验证,并对68例脲原体阳性生殖道标本进行分型和相对定量分析。结果建立的荧光PCR方法对Uu和Up的检测限均为10拷贝,20株Uu和30株Up临床分离株均可正确分型,对22种生殖道道常见微生物无交叉反应。68份脲原体阳性临床标本Uu阳性率为35.3%(24/68),Up阳性率58.8%(40/68),Uu+Up阳性率为5.9%(4/68),分型结果与文献报道的方法一致性为97.1%。标本定量检测显示,Uu阳性标本的中位数为4076拷贝/103细胞,Up阳性标本的中位数为2092拷贝/103细胞。结论建立荧光PCR分型方法可快速完成Uu和Up分型,获得致病型脲原体诊断结果,并通过相对定量反映宿主细胞粘附的病原体量,克服采样差异对检测结果的影响,更客观地反映脲原体载量和致病力。

Objectives To establish a relative quantitative fluorescent PCR technique to detect Ureaplasma urealyticum and Ureaplasma parvum.This novel method does not affect the accuracy of detection results due to sampling differences.Therefore,this method can be used to analyze the biological classification and pathogen load of a Ureaplasma infection in patients.Methods Specific real-time PCR was designed for U.urealyticum and U.parvum,and the humanβ-globin gene(BG)was selected as a quantitative internal standard.Three originally independent real-time PCR methods were combined into a single-tube multiple real-time PCR.Standard curves were plotted using a set of nucleic acid standards.Therefore,the detection limits of U.urealyticum and U.parvum was determined,and the relative content of U.urealyticum and U.parvum relative to the host cell were also effectively calculated.Twenty U.urealyticum clinical isolates,30 U.parvum clinical isolates,and 22 common reproductive tract pathogens were used to verify the sensitivity and specificity of this novel method.Sixty-eight confirmed Ureaplasma-positive genital tract specimens were tested and quantitatively analyzed.Results The lower detection limits of U.urealyticum and U.parvum were both 10 copies.Twenty U.urealyticum and 30 U.parvum clinical isolates were all correctly identified.The novel method did not cross-react with 22 common reproductive tract microorganisms.Sixty-eight 68 Ureaplasma-positive clinical specimens tested positive for U.urealyticum at a rate of 35.3%(24/68),positive for U.parvum at a rate of 58.8%(40/68),and positive for both pathogens at a rate of 5.9%(4/68).The result was 97.1%consistent with methods reported in the literature.Results indicated that the median of U.urealyticum-positive specimens was 4,076 copies/103 cells,and the median of U.parvum-positive specimens was 2,092 copies/103 cells.Conclusion U.urealyticum and U.parvum were identified using a new real-time PCR technique.Host cells were used as a quantitative internal standard to avoid the effects of sampling differences.Therefore,this method indicated the Ureaplasma load and pathogenicity.