南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (12): 1867-1874.doi: 10.12122/j.issn.1673-4254.2022.12.16

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PCR-核酸试纸条快速鉴定皮氏罗尔斯顿菌

王 丹，赵潇颖，姜菲菲，孙丽颖，许 萌，宿建胜，赵云冬   

  1. 北华大学医学技术学院，吉林 吉林 132013
  • 出版日期:2022-12-20 发布日期:2023-01-12

Rapid identification of Ralstonia pickettii using PCR-nucleic acid test strips

WANG Dan, ZHAO Xiaoying, JIANG Feifei, SUN Liying, XU Meng, SU Jiansheng, ZHAO Yundong   

  1. College of Medical Technology, Beihua University, Jilin 132013, China
  • Online:2022-12-20 Published:2023-01-12

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摘要： 目的 基于PCR-核酸试纸条技术，建立快速检测制药用水中皮氏罗尔斯顿菌的方法。方法 利用煮沸法提取皮氏罗尔斯顿菌基因组DNA，以皮氏罗尔斯顿菌16S rDNA为靶基因使用NCBI primer-BLAST 5.0设计一对特异性引物，经克隆转入大肠杆菌感受态细胞DH5α，鉴定PCR产物；分别在引物的5’端标记FITC与Biotin；组装核酸试纸条，建立PCR-核酸试纸条的方法；优化反应体系中胶体金标记的链霉亲和素与抗体浓度的最佳工作量；并对试纸条进行反应原理验证及灵敏度、特异性、稳定性评价；对某生物科技有限公司制药用水检测出的7株皮氏罗尔斯顿菌进行实验并利用MEGA构建进化树，对污染溯源进行分析。结果 煮沸法提取基因组浓度较好，纯度1.8~2.0；PCR产物经克隆转化、测序比对，与GenBank已登记的皮氏罗尔斯顿菌16S rDNA相似度为100%；利用胶体金放大原理，每100 μL胶体金溶液中，加入3.5 μL链霉亲和素进行标记，硝酸纤维素膜上检测线为2.0 mg/mL抗FITC抗体，质控线为1.2 mg/mL生物素化BSA，与阳性扩增产物结合产生红色条带；按照优化条件进行试纸条组装，每100 μL样品展开液中加入8 μL PCR产物，反应5 min后观察结果；核酸试纸条特异性结果与琼脂糖凝胶电泳结果一致，仅有皮氏罗尔斯顿菌为阳性结果，不动杆菌、气单胞菌、假单胞菌、非脱羧勒克氏菌均为阴性结果；核酸试纸条灵敏度评价，将DNA浓度降至10-5 ng/μL时，试纸条检测线仍有条带，较琼脂糖凝胶电泳结果灵敏度高1000倍；核酸试纸条稳定性评价，将试纸条在3、6、9、12月进行检测，稳定性较好。结论 建立PCR-核酸试纸条技术检测制药用水中皮氏罗尔斯顿菌，具有简单快速、特异性强、灵敏度高、成本较低等优点，适用于制药企业对制药用水的日常检测。

关键词: 皮氏罗尔斯顿菌；制药用水；PCR-核酸试纸条；药品；污染菌

Abstract: Objective To develop a method for rapid detection of Ralstonia pickettii in water for pharmaceutical purpose using PCR-nucleic acid test strips. Methods The genomic DNA of Ralstonia pickettii was extracted by boiling method. A pair of specific primers targeting the 16S rDNA with FITC and biotin labeling of the 5' ends was designed and cloned into competent E. coli DH5α cells. The nucleic acid test strips were assembled, and the workload of streptavidin labeled with colloidal gold and antibody concentration in the reaction system was optimized. After verification of the reaction mechanism and assessment of the test sensitivity, specificity and stability, the test strip was used for detecting 7 known strains of Ralstonia pickettii detected in pharmaceutical water, and an evolutionary tree was constructed to analyze the source of contamination. Results The genomic DNA extracted by boiling method had a purity between 1.8 and 2.0, and the PCR products showed a 100% similarity of with Ralstonia pickettii 16S rDNA registered in GenBank. Using the colloidal gold amplification principle, in every 100 μL colloidal gold solution, 3.5 μL streptavidin was added; the detection line on nitrocellulose membrane was 2.0 mg·mL-1 anti FITC antibody, and the quality control line was 1.2 mg · mL- 1 biotinylated BSA, and they generate a red band after binding with positive amplification product. Specificity test of the assembled test strip yielded consistent result with agarose gel electrophoresis without cross reaction with Acinetobacter, Aeromonas, Pseudomonas, or Leclercia adecarboxylata. Sensitivity test of the strip showed a lower detection limit for DNA concentration of 10-5 ng/μL, with a sensitivity 1000 times that of agarose gel electrophoresis. The test strip still had good performance after storage for 3, 6, 9 and 12 months. Conclusion We successfully developed a PCR-nucleic acid test strip for convenient and cost-effective detection of Ralstonia pickettii with good specificity and sensitivity and low cost to facilitate daily monitoring of pharmaceutical water contaminations.

Key words: Ralstonia pickettii; pharmaceutical water; PCR-nucleic acid test strip; drugs; contaminating bacteria