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首页> 美国卫生研究院文献>Journal of Clinical Microbiology >Comparison of Conventional Nested and Real-Time Quantitative PCR for Diagnosis of Scrub Typhus
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Comparison of Conventional Nested and Real-Time Quantitative PCR for Diagnosis of Scrub Typhus

机译：常规巢式和实时定量PCR诊断灌木斑疹伤寒的比较

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Orientia tsutsugamushi is the causative agent of scrub typhus. For the diagnosis of scrub typhus, we investigated the performances of conventional PCR (C-PCR), nested PCR (N-PCR), and real-time quantitative PCR (Q-PCR) targeting the O. tsutsugamushi-specific 47-kDa gene. To compare the detection sensitivities of the three techniques, we used two template systems that used plasmid DNA (plasmid detection sensitivity), including a partial region of the 47-kDa gene, and genomic DNA (genomic detection sensitivity) from a buffy coat sample of a single patient. The plasmid detection sensitivities of C-PCR, N-PCR, and Q-PCR were 5 × 104 copies/μl, 5 copies/μl, and 50 copies/μl, respectively. The results of C-PCR, N-PCR, and Q-PCR performed with undiluted genomic DNA were negative, positive, and positive, respectively. The genomic detection sensitivities of N-PCR and Q-PCR were 64-fold and 16-fold (crossing point [Cp], 37.7; 426 copies/μl), respectively. For relative quantification of O. tsutsugamushi bacteria per volume of whole blood, we performed real-time DNA PCR analysis of the human GAPDH gene, along with the O. tsutsugamushi 47-kDa gene. At a 16-fold dilution, the copy number and genomic equivalent (GE) of GAPDH were 1.1 × 105 copies/μl (Cp, 22.64) and 5.5 × 104 GEs/μl, respectively. Therefore, the relative concentration of O. tsutsugamushi at a 16-fold dilution was 0.0078 organism/one white blood cell (WBC) and 117 organisms/μl of whole blood, because the WBC count of the patient was 1.5 × 104 cells/μl of whole blood. The sensitivities of C-PCR, N-PCR, and Q-PCR performed with blood samples taken from patients within 4 weeks of onset of fever were 7.3% (95% confidence interval [CI], 1.6 to 19.9), 85.4% (95% CI, 70.8 to 94.4), and 82.9% (95% CI, 67.9 to 92.8), respectively. All evaluated assays were 100% specific for O. tsutsugamushi. In conclusion, given its combined sensitivity, specificity, and speed, Q-PCR is the preferred assay for the diagnosis of scrub typhus.
机译：东方tsu虫是灌木斑疹伤寒的病原体。为了诊断斑疹伤寒，我们研究了针对targeting虫专一性47-kDa基因的常规PCR（C-PCR），巢式PCR（N-PCR）和实时定量PCR（Q-PCR）的性能。 。为了比较这三种技术的检测灵敏度，我们使用了两个使用质粒DNA（质粒检测灵敏度）的模板系统，其中包括47-kDa基因的部分区域，以及来自血沉棕黄层样品的基因组DNA（基因组检测灵敏度）。一个病人。 C-PCR，N-PCR和Q-PCR的质粒检测灵敏度分别为5×10 4 拷贝/μl，5拷贝/μl和50拷贝/μl。使用未稀释的基因组DNA进行C-PCR，N-PCR和Q-PCR的结果分别为阴性，阳性和阳性。 N-PCR和Q-PCR的基因组检测灵敏度分别为64倍和16倍（交叉点[Cp]，37.7； 426个拷贝/μl）。为了对全血中volume虫的细菌进行相对定量，我们对human虫的47-kDa基因和人GAPDH基因进行了实时DNA PCR分析。在稀释16倍时，GAPDH的拷贝数和基因组当量（GE）为1.1×10 5 拷贝/μl（Cp，22.64）和5.5×10 4 GEs /μl。因此，由于患者的WBC计数为1.5×10 4，所以16倍稀释度的tsu虫O.的相对浓度为0.0078生物/一个白细胞（WBC）和117生物/μl全血。 细胞/微升全血。在发烧4周内对患者血液样本进行C-PCR，N-PCR和Q-PCR的敏感性分别为7.3％（95％置信区间[CI]，1.6至19.9），85.4％（95） CI分别为70.8到94.4）和82.9％（95％CI为67.9到92.8）。所有评估的测定法都对gam虫O. 100％具有特异性。总之，鉴于其综合的敏感性，特异性和速度，Q-PCR是诊断灌木斑疹伤寒的首选方法。

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