鹦鹉热嗜性衣原体特异性TaqMan-MGB探针荧光定量PCR检测方法的建立与应用

Development and application of specific TaqMan-MGB probe quantitative fluorescence PCR assay for detection on Chlamydophila psittaci

目的建立一种特异、灵敏、重复性好的鹦鹉热嗜性衣原体的TaqMan-MGB荧光定量PCR检测方法。方法利用鹦鹉热嗜性衣原体MOMP基因的特异保守序列设计引物和MGB探针,通过优化,获得最佳反应体系与反应条件,同时进行特异性、重复性、灵敏性评价与Spike-test实验进行临床应用性评价,利用该检测方法对禽鸟类粪便样品进行检测,并与常规PCR检测方法进行比较。结果该方法在0.01pg^100pg范围内线性相关系数为0.999,扩增效率为97.7%;对鹦鹉热嗜性衣原体菌株检测结果均呈现阳性,而非鹦鹉热嗜性衣原体菌株及其它衣原体菌株为阴性;重复性试验中,变异系数为0.317%~0.563%;检测灵敏性为0.01pg;Spike-test试验中最低检出量为25个EB;该方法对禽鸟类粪便样品的检出率为14.3%(40/282),高于常规PCR检测法的7.4%(21/282)。结论本文所建立的Taqman-MGB荧光定量PCR检测法是一种灵敏、高效、稳定,可在嗜性衣原体属中准确检测鹦鹉热嗜性衣原体的方法,该方法的建立更有意于今后对禽鸟类实施鹦鹉热的临床诊断与分子流行病学研究。

In order to develop a specific,sensitive and repeatable quantitative fluorescence PCR technology to detect Chlamydophila psittaci,（C.psittaci）primers and specific TaqMan minor-groove-binding（MGB）hybridization probe were designed for identifying MOMP gene of C.psittaci.Then conditions of PCR reaction were optimized,and the specificity,sensitivity,reproducibility and Spike-test of the assay were evaluated.Besides,the examination results of clinic specimens from birds with both real-time PCR and conventional PCR methods were compared.The method showed a strong linear relationship（R2=0.999）at a range of 0.01 pg-100pg and 97.7% PCR efficiencies;C.psittaci was detected by the assay specifically;the coefficients of variation（CV）value was 0.317%-0.563% and the sensitivity was 0.01pg;the detection limit of the assay in Spike-test was 25 EBs.Otherwise,the positive coincidence rate（14.3%）from this method was higher than that（7.4%）from conventional PCR assay.It is evident that the quantitative fluorescence PCR is a sensitive,efficient,and stable method for the detection of C.psittaci.