联合实时 PCR 和 rpoB 基因焦磷酸测序技术快速鉴定结核分枝杆菌并直接检测临床标本中的利福平耐药性。
Combined real-time PCR and rpoB gene pyrosequencing for rapid identification of Mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens.
机构信息
Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA.
出版信息
J Clin Microbiol. 2010 Apr;48(4):1182-8. doi: 10.1128/JCM.02149-09. Epub 2010 Jan 27.
Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, of both Mycobacterium tuberculosis complex (MTBC) DNA and mutations within the 81-bp core region of the rpoB gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR with internal inhibition assessment and a pyrosequencing assay, was validated for direct use with clinical specimens. To assess the suitability of real-time PCR for use with respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive and AFB-negative specimens, we evaluated specimens received in our laboratory between 11 October 2007 and 30 June 2009. With culture used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values were determined for 1,316 specimens to be as follows: for respiratory specimens, 94.7%, 99.9%, 99.6%, and 98.6%, respectively; for nonrespiratory specimens, 88.5%, 100.0%, 100.0%, and 96.9%, respectively; for AFB-positive specimens, 99.6%, 100.0%, 100.0%, and 97.7%, respectively; and for AFB-negative specimens, 75.4%, 99.9%, 98.0%, and 98.4%, respectively. PCR inhibition was determined to be minimal in this assay, occurring in 0.2% of tests. The rpoB gene pyrosequencing assay was evaluated in a similar prospective study, in which 148 clinical specimens positive for MTBC DNA by real-time PCR were tested. The final results revealed that the results of direct testing of clinical specimens by the pyrosequencing assay were 98.6% concordant with the results of conventional testing for susceptibility to RIF in liquid culture and that our assay displayed adequate sensitivity for 96.6% of the clinical specimens tested. Used together, these assays provide reliable results that aid with the initial management of patients with suspected tuberculosis prior to the availability of the results for cultured material, and they also provide the ability to predict RIF resistance in Mycobacterium tuberculosis-positive specimens in as little as 48 h from the time of clinical specimen receipt.
我们的实验室开发了一种快速、敏感、特异的分子方法，用于在收到临床标本后 48 小时内，检测结核分枝杆菌复合群（MTBC）DNA 以及与利福平（RIF）耐药相关的 rpoB 基因 81 位核心区突变。该方法结合了初始实时 PCR 与内部抑制评估和焦磷酸测序检测，可直接用于临床标本。为了评估实时 PCR 用于呼吸道、非呼吸道、抗酸杆菌（AFB）阳性和 AFB 阴性标本的适用性，我们评估了 2007 年 10 月 11 日至 2009 年 6 月 30 日期间收到的实验室标本。以培养为“金标准”，确定了 1316 个标本的灵敏度、特异性和阳性预测值和阴性预测值分别为：呼吸道标本分别为 94.7%、99.9%、99.6%和 98.6%；非呼吸道标本分别为 88.5%、100.0%、100.0%和 96.9%；AFB 阳性标本分别为 99.6%、100.0%、100.0%和 97.7%；AFB 阴性标本分别为 75.4%、99.9%、98.0%和 98.4%。该检测中 PCR 抑制作用极小，仅 0.2%的检测中出现抑制作用。在一项类似的前瞻性研究中，我们评估了 rpoB 基因焦磷酸测序检测。在该研究中，对实时 PCR 检测 MTBC DNA 阳性的 148 例临床标本进行了直接检测。最终结果显示，焦磷酸测序检测直接检测临床标本的结果与液体培养中对利福平敏感性的常规检测结果的符合率为 98.6%，且我们的检测方法对检测的 96.6%的临床标本具有足够的灵敏度。这两种检测方法联合使用，可在获得培养物结果之前，为疑似结核病患者的初步治疗提供可靠的结果，并能在临床标本送检后 48 小时内预测结核分枝杆菌阳性标本的利福平耐药性。
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