1Microbiology Department (SEALS), St George Hospital, Kogarah 2School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.
Pathology. 2014 Jun;46(4):344-7. doi: 10.1097/PAT.0000000000000098.
The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample inhibition and suitability for detection of low levels of target DNA in random and multiple samples.
本研究的目的是组装和评估针对大肠杆菌丙氨酸消旋酶（alr）基因的非竞争性内部扩增对照（IAC）系统，将其纳入脑膜炎奈瑟菌实时聚合酶链反应（PCR）检测中。设计了针对 IAC 的引物和杂交探针，并评估了其特异性。使用脑膜炎奈瑟菌在盐水中、混合脑脊髓液（CSF）和 EDTA 血液中的系列稀释液构建标准曲线，推断出组装检测的扩增效率和检测限。计算了脑膜炎奈瑟菌 ctrA 在盐水中（阴性空白）和脑膜炎奈瑟菌阴性 CSF 和 EDTA 血液样本的 IAC 交叉点的 95%置信区间（CI）。这些限值作为参考范围，用于比较前瞻性检测中记录的 IAC 交叉点，以检测样品抑制。该系统用于测试两例脑膜炎球菌病患者的连续 EDTA 血液样本。IAC 系统特异性针对大肠杆菌和志贺氏菌。包含 IAC 系统不会影响组装检测中脑膜炎奈瑟菌的扩增效率和检测低靶 DNA 水平的能力。IAC 交叉点在 CSF 和 EDTA 血液的临床样本中有所不同。阐明的 EDTA 血液参考范围用于检测两个临床病例之一中的样品抑制。IAC 系统监测组装的脑膜炎奈瑟菌检测中所有过程的性能。测量 IAC 交叉点可作为样品稳定性和抑制特性的指标，用于测试来自同一患者的单个或多个样本。对大肠杆菌和志贺氏菌的特异性使它能够纳入同一实验室中不同靶标的检测。在 IAC 交叉点和参考范围的背景下报告 PCR 检测结果可验证样品抑制和检测随机和多个样本中低水平靶 DNA 的适用性。
