基于互补锁式引物的实时 PCR 方法增强对柯克斯体的检测。

Enhanced detection of Coxiella burnetii with a complementary locked primer-based real-time PCR method.

机构信息

Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control Prevention, Chungcheongbuk-do, Korea.

出版信息

Mol Diagn Ther. 2011 Apr 1;15(2):103-7. doi: 10.1007/BF03256399.

DOI:10.1007/BF03256399

PMID:21452906

Abstract

BACKGROUND AND OBJECTIVE

Coxiella burnetii is the bacterial causative agent of Q fever in humans. Because Q fever can establish itself with an initial inoculation of fewer than ten C. burnetii cells, a sensitive detection method for C. burnetii infection is needed for early detection. We aimed to evaluate the effectiveness of a complementary locked primer (CLP)-based real-time PCR method for sensitive detection of C. burnetii infection.

METHODS

To evaluate the ability of CLPs to enhance the efficiency of the real-time PCR assay for the C. burnetii IS1111 insertion sequence, the mean threshold cycle values from 20 real-time PCR replicates with either CLPs or conventional primers were determined using tenfold serial dilutions (102-108) of purified C. burnetii Nine Mile genomic DNA. In addition, the cross-reactivity between C. burnetii and 31 non-Coxiella species was examined.

RESULTS

The CLP-based real-time PCR allowed specific and reliable detection of as few as 59 copies of the IS1111 element present in the genome of C. burnetii, which represents approximately 2.96 genome equivalents or three cells of C. burnetii. These results demonstrate the effectiveness of CLP-based real-time PCR for sensitive detection of C. burnetii infection.

CONCLUSION

It can be concluded that the CLP-based real-time PCR assay is a more appropriate method for sensitive detection and quantification of C. burnetii than previously reported methods.

摘要

背景与目的

柯克斯体是人类贝氏柯克斯体病的细菌病原体。由于 Q 热可以通过初始接种少于 10 个贝氏柯克斯体细胞而建立，因此需要一种用于早期检测的贝氏柯克斯体感染的敏感检测方法。我们旨在评估基于互补锁定引物（CLP）的实时 PCR 方法对贝氏柯克斯体感染的检测效果。

方法

为了评估 CLP 对提高基于实时 PCR 检测贝氏柯克斯体 IS1111 插入序列效率的能力，使用 10 倍系列稀释（102-108）的纯化贝氏柯克斯体 Nine Mile 基因组 DNA，通过 20 次实时 PCR 重复实验确定使用 CLP 或常规引物的平均阈值循环值。此外，还检测了 CLP 与 31 种非柯克斯体物种之间的交叉反应性。