Two Multiplex Real-Time PCR Assays to Detect and Differentiate <i>Acinetobacter baumannii</i> and Non- <i>baumannii Acinetobacter</i> spp. Carrying <i>bla</i>_NDM, <i>bla</i>_OXA-23-Like, <i>bla</i>_OXA-40-Like, <i>bla</i>_OXA-51-Like, and <i>bla</i>_OXA-58-Like Genes

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.全球范围内，由耐碳青霉烯类不动杆菌属（Acinetobacter spp.）引发的医院感染报道日益增多。耐碳青霉烯类不动杆菌（Carbapenem-resistant Acinetobacter, CRA）正成为严重的公共卫生隐患，患者的发病率、死亡率以及住院时长均随之升高。因此，快速检测CRA对于流行病学监测至关重要。聚合酶链式反应（Polymerase chain reaction, PCR）已被广泛用于多数病原菌的快速鉴定。本研究开发了两种多重实时荧光定量PCR（multiplex real-time PCR）检测方法，用于检测并区分鲍曼不动杆菌（Acinetobacter baumannii, A. baumannii）与非鲍曼不动杆菌属不动杆菌，以及常见的碳青霉烯酶基因，包括blaNDM、blaOXA-23-like、blaOXA-40-like、blaOXA-51-like及blaOXA-58-like。本研究证实，上述检测方法可直接在临床标本中检测blaNDM阳性、blaOXA-23-like阳性、blaOXA-40-like阳性、blaOXA-51-like阳性及blaOXA-58-like阳性的CRA，具备潜在应用价值。研究人员特异性设计了引物，开发出两种多重实时荧光定量PCR检测方法：检测方法1用于检测鲍曼不动杆菌16S–23S rRNA内部转录间隔区序列、不动杆菌recA基因以及B类金属酶编码基因blaNDM；检测方法2则用于检测D类氧头孢烯酶编码基因（blaOXA-23-like、blaOXA-40-like、blaOXA-51-like及blaOXA-58-like）。所有检测均在ABI Prism 7500 FAST实时荧光定量PCR系统上完成。以CRA分离株为样本，将本研究建立的检测方法与常规PCR及测序技术进行对比验证。将定量的CRA菌体添加至痰液及粪便标本中，用于验证该多重实时荧光定量PCR检测方法的性能。靶标与非靶标扩增结果显示，该多重实时荧光定量PCR检测方法特异性良好；各靶标的检测限为每20 μL反应体系10个拷贝；靶标基因在6个对数级稀释梯度内均呈现良好线性关系（决定系数r²>0.99）；批内与批间重复性的Ct值变异系数均小于5%。在对400株CRA分离株进行检测时，该多重实时荧光定量PCR检测方法与常规PCR的符合率达100%；在痰液及粪便标本中，其对靶标DNA的检测灵敏度为10² CFU/mL。综上，本研究开发的新型多重实时荧光定量PCR检测方法可灵敏且特异地鉴定并区分blaNDM阳性、blaOXA-23-like阳性、blaOXA-40-like阳性、blaOXA-51-like阳性及blaOXA-58-like阳性的CRA，有望成为直接检测临床标本中CRA以及开展医院感染监测的潜在工具。