实时荧光定量PCR技术的优化及其在恙虫病东方体诊断中的应用

目的建立一种优化的实时荧光定量PCR技术,并用于诊断粤北山区恙虫病东方体(Ot)基因型。方法采用优化实时荧光定量PCR技术对感染Ot的患者和鼠类的血液和组织标本检测Ot DNA载量并分析其基因型,并与未优化实时荧光定量PCR和巢式PCR的检测结果进行比较。结果采用优化实时荧光定量PCR技术检测660例发热患者,有224例检测到Ot,其中108例是发热5d内的早期患恙虫病患者,以及在55份鼠类的标本中有12份能检测到感染Ot,均为Gilliam型;未优化实时荧光定量PCR在患病7d以上才能检测到Ot。同时,在鼠类基因扩增片段还显示出与Gilliam、Karp、Kato3株国际参考株不同,经巢式PCR分型证实为Kawasaki型,与巢式PCR基因分型的结果相符。结论优化实时荧光定量PCR技术可作为早期诊断恙虫病的实验方法,用于快速分析Ot基因型,适合在基层医院推广应用。

Application of optimized real-time fluorescent quantitation PCR for the early diagnosis of Orientia tsutsugamushi

Objective To construct an optimized real-time fluorescent quantitation polymerase chain reaction(qPCR) assay for the detection of Orientia tsutsugamushi(Ot).Methods DNA concentration and genotype of Ot were detected by optimized real-time qPCR assay in blood and tissue samples from patients and mice with infection of Ot,and the detection results were compared with that detected by un-optimized real-time qPCR and nest PCR(nPCR).Results 660 patients with fever were detected by real-time qPCR,and 224 cases were confirmed to be with infection of Ot,including 108 patients were at early stage of akamushi disease and with fever less than 5 days,and in 55 samples from mice,12 cases were positive with Ot for Gilliam strains.Ot could be detected by un-optimized real-time qPCR in patients with fever for at least 7 days,and samples from mice could be detected with gene fragment different from reference strains of Gilliam,Karp and Kato,which could be confirmed to be Kawasaki type by nPCR,identical to that acquired by nPCR.Conclusion Optimized real-time qPCR assay could be a rapid,simple and accurate method for diagnosis of patients with akamushi disease.