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首页> 外文期刊>Journal of Clinical Microbiology >Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens
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Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

机译：靶向hsp65基因的多探针实时PCR方法的开发和应用

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We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.
机译：我们开发了一种针对 hsp65 （HMPRT-PCR）的多探针实时PCR检测方法，以检测和鉴定分枝杆菌分离株以及直接从痰标本中分离出来的分离株。根据 hsp65 基因序列设计了用于HMPRT-PCR的引物和探针，可识别7种致病分枝杆菌，包括结核分枝杆菌， M。 avium ， M。细胞内， M。堪萨斯州， M。脓肿， M。 massiliense 和 M。 该技术已应用于24例参比菌株和133株临床分离株，并以100％的敏感性和特异性在所有菌株之间进行了区分。此外，该方法还应用于连续117例涂片阳性患者的痰标本，涂片结果从痕量到3+。然后将这些结果与使用 rpoB PCR限制性分析方法从相同痰标本培养物中获得的结果进行比较。 HMPRT-PCR方法正确鉴定了89个样品中的分枝杆菌（76.0％，89/117），此外，耐酸芽孢杆菌得分等于或更高的痰液的敏感性水平提高到94.3％（50/53）。超过2岁以上。我们的数据表明，这种新颖的HMPRT-PCR方法可能是一种有前途的方法，可用于从痰液样本和常规培养物中分离出的细菌分离物中检测病原性分枝杆菌。

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