USP一般章节<60>用于检测非无菌产品中的洋葱伯克霍尔德氏菌(Bcc)成员于2019年12月正式生效。这种分离方法需要通过额外的鉴定测试(参考解释部分)来确认在洋葱伯克霍尔德氏菌选择性琼脂(BCSA)上发现的任何生长的身份。本文提出了一种单重PCR方法,可快速确认Bcc组中在BCSA(和其他营养培养基)上生长的任何微生物的成员资格。该方法具有成本效益,因为它不需要昂贵的设备或试剂;因此,它可以很容易地在行业中采用。我们使用先前发布的PCR引物和37个临床和环境Bcc分离株的扩展组验证了这种单重PCRBcc鉴定方法。这些Bcc分离物的来源和存储库包括受污染的健康产品和医疗设备,感染囊性纤维化的患者,国家微生物学实验室(NML)内部银行和美国类型培养收集(ATCC)。使用我们的确证鉴定方法,所有属于Bcc的37个分离株都已检测为阳性。22个阴性对照,包括4个属于伯克霍尔德氏菌属的分离株,如预期的那样被测试为阴性。我们的工作表明,这种单重PCR是一种有效的Bcc鉴定验证方法,它可以成功地补充USP章节<60>在药品中发现的Bcc分离株鉴定。
United States Pharmacopeia (USP) General Chapter <60> for the detection of Burkholderia cepacia complex (Bcc) members in nonsterile products became official in December 2019. This isolation method requires confirmation of the identity of any growth found on Burkholderia cepacia Selective Agar (BCSA) by additional identification tests (refer to the Interpretation section). This article presents a singleplex polymerase chain reaction (PCR) method to rapidly confirm the membership of any microbial grown on BCSA (and other nutrient medium) in the Bcc group. This method is cost effective as it does not require expensive equipment or reagents; therefore, it can be easily adopted in the industry without an important investment. We validated this singleplex PCR Bcc identification method with previously published PCR primers with an expanded panel of 37 clinical and environmental Bcc isolates. The sources and repositories of these Bcc isolates include contaminated health products and medical devices, patients infected with cystic fibrosis, the National Microbiology Laboratory (NML) internal strain bank, and the American Type Culture Collection (ATCC). All 37 isolates that belong to the Bcc tested positive using our confirmatory identification method. Twenty-two negative controls including four isolates belonging to the genus Burkholderia tested negative as expected. Our work indicates that this singleplex PCR is an efficient confirmatory method for Bcc identification, and it can successfully supplement USP <60> for Bcc isolates identification found in pharmaceutical products.